Detection of colorectal cancer

ABSTRACT

The present disclosure provides, among other things, methods for colorectal cancer detection (e.g., screening) and compositions related thereto. In various embodiments, the present disclosure provides methods for colorectal cancer screening that include analysis of methylation status of one or more methylation biomarkers, and compositions related thereto. In various embodiments, the present disclosure provides methods for colorectal cancer detection (e.g., screening) that include detecting (e.g., screening) methylation status of one or more methylation biomarkers in cfDNA, e.g., in ctDNA. In various embodiments, the present disclosure provides methods for colorectal cancer screening that include detecting (e.g., screening) methylation status of one or more methylation biomarkers in cfDNA, e.g., in ctDNA, using MSRE-qPCR.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. application Ser. No.16/428,865 filed on May 31, 2019, and U.S. Provisional Application No.62/956,059, filed Dec. 31, 2019, the disclosure of each of which ishereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy is named 2011722_0045_SL.txt and is2,771,268 bytes in size.

BACKGROUND

Cancer screening is a critical component of cancer prevention,diagnosis, and treatment. Colorectal cancer (CRC) has been identified,according to some reports, as the third most common type of cancer andthe second most frequent cause of cancer mortality in the world.According to some reports, there are over 1.8 million new cases ofcolorectal cancer per year and about 881,000 deaths from colorectalcancer, accounting for about 1 in 10 cancer deaths. Regular colorectalcancer screening is recommended, particular for individuals over age 50.Moreover, incidence of colorectal cancer in individuals below 50 hasincreased over time. Statistics suggest that current colorectal cancerscreening techniques are insufficient.

SUMMARY

Despite improvements over time, only about 40-44% of colorectal cancersare currently detected by screening in an early, localized stage. Thisis at least in part due to insufficient sensitivity and/or specificityof current screening techniques. Currently recommended techniquesinclude colonoscopy and/or fecal blood testing for those over age 50.

The present disclosure provides, among other things, methods forcolorectal cancer screening and compositions related thereto. In variousembodiments, the present disclosure provides methods for colorectalcancer screening that include determination of methylation status (e.g.,the number, frequency, or pattern of methylation) at one or moremethylation sites found within a methylation locus, e.g., adifferentially methylated region (DMR), of deoxyribonucleic acid (DNA)of a human subject, and compositions related thereto. In variousembodiments, the present disclosure provides methods for colorectalcancer screening that include screening methylation status for each ofone or more methylation loci in cfDNA (cell free DNA), e.g., in ctDNA(circulating tumor DNA). In various embodiments, the present disclosureprovides methods for colorectal cancer screening that includedetermining a methylation status for each of one or more methylationloci in cfDNA, e.g., in ctDNA, using quantitative polymerase chainreaction (qPCR) (e.g., methylation sensitive restriction enzymequantitative polymerase chain reaction, MSRE-qPCR). Various compositionsand methods provided herein provide sensitivity and specificitysufficient for clinical application in colorectal cancer screening.Various compositions and methods provided herein are useful incolorectal cancer screening by analysis of an accessible tissue sampleof a subject, e.g., a tissue sample that is blood or a blood component(e.g., cfDNA, e.g., ctDNA), or stool.

In one aspect, the invention is directed to a method of detecting (e.g.,screening for) colorectal cancer, the method comprising: determining amethylation status [e.g., a number, frequency, or pattern of methylationat one or more methylation sites within a methylation locus] for each ofthe following, in deoxyribonucleic acid (DNA) of a human subject: (a) amethylation locus [e.g., a differentially methylated region] within geneZNF132; (b) a first methylation locus within gene ADAMTS2; and (c) asecond methylation locus within gene ADAMTS2; and diagnosing colorectalcancer in the human subject based on said determined methylationstatuses.

In certain embodiments, the method further comprises determining amethylation status for each of the following, in the DNA of the humansubject: (d) a methylation locus within gene ZNF542; and (e) amethylation locus within gene LONRF2.

In certain embodiments, the method further comprises determining amethylation status for a methylation locus within gene ZNF492 in the DNAof the human subject.

In certain embodiments, the methylation locus within gene ZNF132comprises ZNF132 '415 (SEQ ID NO: 40).

In certain embodiments, the first methylation locus within gene ADAMTS2comprises ADAMTS2 '254 (SEQ ID NO: 21).

In certain embodiments, the second methylation locus within gene ADAMTS2comprises ADAMTS2 '284 (SEQ ID NO: 22).

In certain embodiments, the methylation locus within gene ZNF542comprises ZNF542 '502 (SEQ ID NO: 35).

In certain embodiments, the methylation locus within gene LONRF2comprises LONRF2 '281 (SEQ ID NO: 19).

In certain embodiments, the methylation locus within gene ZNF492comprises ZNF492 '069 (SEQ ID NO: 42).

In certain embodiments, the DNA is isolated from blood or plasma of thehuman subject.

In certain embodiments, the DNA is cell-free DNA of the human subject.

In certain embodiments, methylation status is determined usingquantitative polymerase chain reaction (qPCR) (e.g., methylationsensitive restriction enzyme quantitative polymerase chain reaction,MSRE-qPCR).

In another aspect, the invention is directed to a kit for use incolorectal cancer detection (e.g., screening), the kit comprising: (a)an oligonucleotide primer pair for amplification of a methylation locuswithin gene ZNF132; (b) an oligonucleotide primer pair for amplificationof a first methylation locus within gene ADAMTS2; and (c) anoligonucleotide primer pair for amplification of a second methylationlocus within gene ADAMTS2 (e.g., and, optionally, the kit furthercomprising at least one methylation sensitive restriction enzyme).

In certain embodiments, the kit further comprises: (d) anoligonucleotide primer pair for amplification of a methylation locuswithin gene ZNF542; and (e) an oligonucleotide primer pair foramplification of a methylation locus within gene LONRF2.

In certain embodiments, the kit further comprises: (f) anoligonucleotide primer pair for amplification of a methylation locuswithin gene ZNF492.

In certain embodiments, (a) is an oligonucleotide primer pair foramplification of ZNF132 '415 (primer pair SEQ ID NO: 91 and SEQ ID NO:92).

In certain embodiments, (b) is an oligonucleotide primer pair foramplification of ADAMTS2 '254 (primer pair SEQ ID NO: 53 and SEQ ID NO:54).

In certain embodiments, (c) is an oligonucleotide primer pair foramplification of ADAMTS2 '284 (primer pair SEQ ID NO: 55 and SEQ ID NO:56).

In certain embodiments, (d) is an oligonucleotide primer pair foramplification of ZNF542 '502 (primer pair SEQ ID NO: 81 and SEQ ID NO:82).

In certain embodiments, (e) is an oligonucleotide primer pair foramplification of LONRF2 '281 (primer pair SEQ ID NO: 49 and SEQ ID NO:50).

In certain embodiments, (f) is an oligonucleotide primer pair foramplification of ZNF492 '069 (primer pair SEQ ID NO: 95 and SEQ ID NO:96).

In another aspect, the invention is directed to a diagnostic qPCRreaction for detection (e.g., screening) of colorectal cancer, thediagnostic qPCR reaction including: (a) human DNA; (b) a polymerase; (c)an oligonucleotide primer pair for amplification of a methylation locuswithin gene ZNF132; (d) an oligonucleotide primer pair for amplificationof a first methylation locus within gene ADAMTS2; (e) an oligonucleotideprimer pair for amplification of a second methylation locus within geneADAMTS2; and (f) optionally, at least one methylation sensitiverestriction enzyme.

In certain embodiments, the reaction further comprises: (g) anoligonucleotide primer pair for amplification of a methylation locuswithin gene ZNF542; and (h) an oligonucleotide primer pair foramplification of a methylation locus within gene LONRF2.

In certain embodiments, the reaction further comprises: (i) anoligonucleotide primer pair for amplification of a methylation locuswithin gene ZNF492.

In certain embodiments, (c) is an oligonucleotide primer pair foramplification of ZNF132 '415 (primer pair SEQ ID NO: 91 and SEQ ID NO:92).

In certain embodiments, (d) is an oligonucleotide primer pair foramplification of ADAMTS2 '254 (primer pair SEQ ID NO: 53 and SEQ ID NO:54).

In certain embodiments, (e) is an oligonucleotide primer pair foramplification of ADAMTS2 '284 (primer pair SEQ ID NO: 55 and SEQ ID NO:56).

In certain embodiments, (g) is an oligonucleotide primer pair foramplification of ZNF542 '502 (primer pair SEQ ID NO: 81 and SEQ ID NO:82).

In certain embodiments, (h) is an oligonucleotide primer pair foramplification of LONRF2 '281 (primer pair SEQ ID NO: 49 and SEQ ID NO:50).

In certain embodiments, (i) is an oligonucleotide primer pair foramplification of ZNF492 '069 (primer pair SEQ ID NO: 95 and SEQ ID NO:96).

In various aspects, methods and compositions of the present inventioncan be used in combination with biomarkers known in the art, e.g., asdisclosed in U.S. Pat. No. 10,006,925, which is herein incorporated byreference in its entirety.

Definitions

A or An: The articles “a” and “an” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” refers to one element or morethan one element.

About: The term “about”, when used herein in reference to a value,refers to a value that is similar, in context, to the referenced value.In general, those skilled in the art, familiar with the context, willappreciate the relevant degree of variance encompassed by “about” inthat context. For example, in some embodiments, e.g., as set forthherein, the term “about” can encompass a range of values that within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or with a fraction of a percent, of the referredvalue.

Administration: As used herein, the term “administration” typicallyrefers to the administration of a composition to a subject or system,for example to achieve delivery of an agent that is, is included in, oris otherwise delivered by, the composition.

Agent: As used herein, the term “agent” refers to an entity (e.g., forexample, a small molecule, peptide, polypeptide, nucleic acid, lipid,polysaccharide, complex, combination, mixture, system, or phenomenonsuch as heat, electric current, electric field, magnetic force, magneticfield, etc.).

Amelioration: As used herein, the term “amelioration” refers to theprevention, reduction, palliation, or improvement of a state of asubject. Amelioration includes, but does not require, complete recoveryor complete prevention of a disease, disorder or condition.

Amplicon or amplicon molecule: As used herein, the term “amplicon” or“amplicon molecule” refers to a nucleic acid molecule generated bytranscription from a template nucleic acid molecule, or a nucleic acidmolecule having a sequence complementary thereto, or a double-strandednucleic acid including any such nucleic acid molecule. Transcription canbe initiated from a primer.

Amplification: As used herein, the term “amplification” refers to theuse of a template nucleic acid molecule in combination with variousreagents to generate further nucleic acid molecules from the templatenucleic acid molecule, which further nucleic acid molecules may beidentical to or similar to (e.g., at least 70% identical, e.g., at least75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to) a segment of the template nucleic acid molecule and/or asequence complementary thereto.

Amplification reaction mixture: As used herein, the terms “amplificationreaction mixture” or “amplification reaction” refer to a templatenucleic acid molecule together with reagents sufficient foramplification of the template nucleic acid molecule.

Biological Sample: As used herein, the term “biological sample”typically refers to a sample obtained or derived from a biologicalsource (e.g., a tissue or organism or cell culture) of interest, asdescribed herein. In some embodiments, e.g., as set forth herein, abiological source is or includes an organism, such as an animal orhuman. In some embodiments, e.g., as set forth herein, a biologicalsample is or include biological tissue or fluid. In some embodiments,e.g., as set forth herein, a biological sample can be or include cells,tissue, or bodily fluid. In some embodiments, e.g., as set forth herein,a biological sample can be or include blood, blood cells, cell-free DNA,free floating nucleic acids, ascites, biopsy samples, surgicalspecimens, cell-containing body fluids, sputum, saliva, feces, urine,cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph,gynecological fluids, secretions, excretions, skin swabs, vaginal swabs,oral swabs, nasal swabs, washings or lavages such as a ductal lavages orbroncheoalveolar lavages, aspirates, scrapings, bone marrow. In someembodiments, e.g., as set forth herein, a biological sample is orincludes cells obtained from a single subject or from a plurality ofsubjects. A sample can be a “primary sample” obtained directly from abiological source, or can be a “processed sample.” A biological samplecan also be referred to as a “sample.”

Biomarker: As used herein, the term “biomarker,” consistent with its usein the art, refers to a to an entity whose presence, level, or form,correlates with a particular biological event or state of interest, sothat it is considered to be a “marker” of that event or state. Those ofskill in the art will appreciate, for instance, in the context of a DNAbiomarker, that a biomarker can be or include a locus (such as one ormore methylation loci) and/or the status of a locus (e.g., the status ofone or more methylation loci). To give but a few examples of biomarkers,in some embodiments, e.g., as set forth herein, a biomarker can be orinclude a marker for a particular disease, disorder or condition, or canbe a marker for qualitative of quantitative probability that aparticular disease, disorder or condition can develop, occur, orreoccur, e.g., in a subject. In some embodiments, e.g., as set forthherein, a biomarker can be or include a marker for a particulartherapeutic outcome, or qualitative of quantitative probability thereof.Thus, in various embodiments, e.g., as set forth herein, a biomarker canbe predictive, prognostic, and/or diagnostic, of the relevant biologicalevent or state of interest. A biomarker can be an entity of any chemicalclass. For example, in some embodiments, e.g., as set forth herein, abiomarker can be or include a nucleic acid, a polypeptide, a lipid, acarbohydrate, a small molecule, an inorganic agent (e.g., a metal orion), or a combination thereof. In some embodiments, e.g., as set forthherein, a biomarker is a cell surface marker. In some embodiments, e.g.,as set forth herein, a biomarker is intracellular. In some embodiments,e.g., as set forth herein, a biomarker is found outside of cells (e.g.,is secreted or is otherwise generated or present outside of cells, e.g.,in a body fluid such as blood, urine, tears, saliva, cerebrospinalfluid, and the like). In some embodiments, e.g., as set forth herein, abiomarker is methylation status of a methylation locus. In someinstances, e.g., as set forth herein, a biomarker may be referred to asa “marker.”

To give but one example of a biomarker, in some embodiments e.g., as setforth herein, the term refers to expression of a product encoded by agene, expression of which is characteristic of a particular tumor, tumorsubclass, stage of tumor, etc. Alternatively or additionally, in someembodiments, e.g., as set forth herein, presence or level of aparticular marker can correlate with activity (or activity level) of aparticular signaling pathway, for example, of a signaling pathway theactivity of which is characteristic of a particular class of tumors.

Those of skill in the art will appreciate that a biomarker may beindividually determinative of a particular biological event or state ofinterest, or may represent or contribute to a determination of thestatistical probability of a particular biological event or state ofinterest. Those of skill in the art will appreciate that markers maydiffer in their specificity and/or sensitivity as related to aparticular biological event or state of interest.

Blood component: As used herein, the term “blood component” refers toany component of whole blood, including red blood cells, white bloodcells, plasma, platelets, endothelial cells, mesothelial cells,epithelial cells, and cell-free DNA. Blood components also include thecomponents of plasma, including proteins, metabolites, lipids, nucleicacids, and carbohydrates, and any other cells that can be present inblood, e.g., due to pregnancy, organ transplant, infection, injury, ordisease.

Cancer: As used herein, the terms “cancer,” “malignancy,” “neoplasm,”“tumor,” and “carcinoma,” are used interchangeably to refer to adisease, disorder, or condition in which cells exhibit or exhibitedrelatively abnormal, uncontrolled, and/or autonomous growth, so thatthey display or displayed an abnormally elevated proliferation rateand/or aberrant growth phenotype. In some embodiments, e.g., as setforth herein, a cancer can include one or more tumors. In someembodiments e.g., as set forth herein, a cancer can be or include cellsthat are precancerous (e.g., benign), malignant, pre-metastatic,metastatic, and/or non-metastatic. In some embodiments e.g., as setforth herein, a cancer can be or include a solid tumor. In someembodiments e.g., as set forth herein, a cancer can be or include ahematologic tumor. In general, examples of different types of cancersknown in the art include, for example, colorectal cancer, hematopoieticcancers including leukemias, lymphomas (Hodgkin's and non-Hodgkin's),myelomas and myeloproliferative disorders; sarcomas, melanomas,adenomas, carcinomas of solid tissue, squamous cell carcinomas of themouth, throat, larynx, and lung, liver cancer, genitourinary cancerssuch as prostate, cervical, bladder, uterine, and endometrial cancer andrenal cell carcinomas, bone cancer, pancreatic cancer, skin cancer,cutaneous or intraocular melanoma, cancer of the endocrine system,cancer of the thyroid gland, cancer of the parathyroid gland, head andneck cancers, breast cancer, gastro-intestinal cancers and nervoussystem cancers, benign lesions such as papillomas, and the like.

Chemotherapeutic agent: As used herein, the term “chemotherapeuticagent,” consistent with its use in the art, refers to one or more agentsknown, or having characteristics known to, treat or contribute to thetreatment of cancer. In particular, chemotherapeutic agents includepro-apoptotic, cytostatic, and/or cytotoxic agents. In some embodimentse.g., as set forth herein, a chemotherapeutic agent can be or includealkylating agents, anthracyclines, cytoskeletal disruptors (e.g.,microtubule targeting moieties such as taxanes, maytansine, and analogsthereof, of), epothilones, histone deacetylase inhibitors HDACs),topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and/ortopoisomerase II), kinase inhibitors, nucleotide analogs or nucleotideprecursor analogs, peptide antibiotics, platinum-based agents,retinoids, vinca alkaloids, and/or analogs that share a relevantanti-proliferative activity. In some particular embodiments e.g., as setforth herein, a chemotherapeutic agent can be or include of Actinomycin,All-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine,Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin,Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin,Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone,Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib,Irinotecan, Maytansine and/or analogs thereof (e.g., DM1)Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, aMaytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide,Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine,Vinorelbine, or a combination thereof. In some embodiments e.g., as setforth herein, a chemotherapeutic agent can be utilized in the context ofan antibody-drug conjugate. In some embodiments e.g., as set forthherein, a chemotherapeutic agent is one found in an antibody-drugconjugate selected from the group consisting of: hLL1-doxorubicin,hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38,hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox,hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox,P4/D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin,trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin,SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595,BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMAADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600,RG-7636, ABT-414, IMGN-853, IMGN-529, vorsetuzumab mafodotin, andlorvotuzumab mertansine. In some embodiments e.g., as set forth herein,a chemotherapeutic agent can be or comprise of farnesyl-thiosalicylicacid (FTS), 4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH),estradiol (E2), tetramethoxystilbene (TMS), δ-tocatrienol, salinomycin,or curcumin.

Combination therapy: As used herein, the term “combination therapy”refers to administration to a subject of to two or more agents orregimens such that the two or more agents or regimens together treat adisease, condition, or disorder of the subject. In some embodiments,e.g., as set forth herein, the two or more therapeutic agents orregimens can be administered simultaneously, sequentially, or inoverlapping dosing regimens. Those of skill in the art will appreciatethat combination therapy includes but does not require that the twoagents or regimens be administered together in a single composition, norat the same time.

Comparable: As used herein, the term “comparable” refers to memberswithin sets of two or more conditions, circumstances, agents, entities,populations, etc., that may not be identical to one another but that aresufficiently similar to permit comparison there between, such that oneof skill in the art will appreciate that conclusions can reasonably bedrawn based on differences or similarities observed. In someembodiments, e.g., as sort forth herein, comparable sets of conditions,circumstances, agents, entities, populations, etc. are typicallycharacterized by a plurality of substantially identical features andzero, one, or a plurality of differing features. Those of ordinary skillin the art will understand, in context, what degree of identity isrequired to render members of a set comparable. For example, those ofordinary skill in the art will appreciate that members of sets ofconditions, circumstances, agents, entities, populations, etc., arecomparable to one another when characterized by a sufficient number andtype of substantially identical features to warrant a reasonableconclusion that differences observed can be attributed in whole or partto non-identical features thereof.

Detectable moiety: The term “detectable moiety” as used herein refers toany element, molecule, functional group, compound, fragment, or othermoiety that is detectable. In some embodiments, e.g., as sort forthherein, a detectable moiety is provided or utilized alone. In someembodiments, e.g., as sort forth herein, a detectable moiety is providedand/or utilized in association with (e.g., joined to) another agent.Examples of detectable moieties include, but are not limited to, variousligands, radionuclides (e.g., ³H, ¹⁴C, ¹⁸F, ¹⁹F, ³²P, ³⁵S, ¹³⁵I, ¹²⁵I,¹²³I, ⁶⁴Cu, ¹⁸⁷Re, ¹¹¹In, ⁹⁰Y, ^(99m)Tc, ¹⁷⁷Lu, ⁸⁹Zr etc.), fluorescentdyes, chemiluminescent agents, bioluminescent agents, spectrallyresolvable inorganic fluorescent semiconductors nanocrystals (i.e.,quantum dots), metal nanoparticles, nanoclusters, paramagnetic metalions, enzymes, colorimetric labels, biotin, dioxigenin, haptens, andproteins for which antisera or monoclonal antibodies are available.

Diagnosis: As used herein, the term “Diagnosis” refers to determiningwhether, and/or the qualitative of quantitative probability that, asubject has or will develop a disease, disorder, condition, or state.For example, in diagnosis of cancer, diagnosis can include adetermination regarding the risk, type, stage, malignancy, or otherclassification of a cancer. In some instances, e.g., as sort forthherein, a diagnosis can be or include a determination relating toprognosis and/or likely response to one or more general or particulartherapeutic agents or regimens.

Diagnostic information: As used herein, the term “diagnosticinformation” refers to information useful in providing a diagnosis.Diagnostic information can include, without limitation, biomarker statusinformation.

Differentially methylated: As used herein, the term “differentiallymethylated” describes a methylation site for which the methylationstatus differs between a first condition and a second condition. Amethylation site that is differentially methylated can be referred to asa differentially methylated site. In some instances, e.g., as sort forthherein, a DMR is defined by the amplicon produced by amplification usingoligonucleotide primers, e.g., a pair of oligonucleotide primersselected for amplification of the DMR or for amplification of a DNAregion of interest present in the amplicon. In some instances, e.g., assort forth herein, a DMR is defined as a DNA region amplified by a pairof oligonucleotide primers, including the region having the sequence of,or a sequence complementary to, the oligonucleotide primers. In someinstances, e.g., as sort forth herein, a DMR is defined as a DNA regionamplified by a pair of oligonucleotide primers, excluding the regionhaving the sequence of, or a sequence complementary to, theoligonucleotide primers. As used herein, a specifically provided DMR canbe unambiguously identified by the name of an associated gene followedby three digits of a starting position, such that, for example, a DMRstarting at position 29921434 of ALK can be identified as ALK '434.

Differentially methylated region: As used herein, the term“differentially methylated region” (DMR) refers to a DNA region thatincludes one or more differentially methylated sites. A DMR thatincludes a greater number or frequency of methylated sites under aselected condition of interest, such as a cancerous state, can bereferred to as a hypermethylation DMR. A DMR that includes a smallernumber or frequency of methylated sites under a selected condition ofinterest, such as a cancerous state, can be referred to as ahypomethylation DMR. A DMR that is a methylation biomarker forcolorectal cancer can be referred to as a colorectal cancer DMR. In someinstances, e.g., as set forth herein, a DMR can be a single nucleotide,which single nucleotide is a methylation site. In some instances, e.g.,as set forth herein, a DMR has a length of at least 10, at least 15, atleast 20, at least 24, at least 50, or at least 75 base pairs. In someinstances, e.g., as set forth herein, a DMR has a length of less than1000, less than 750, less than 500, less than 350, less than 300, orless than 250 base pairs (e.g., where methylation status is determinedusing quantitative polymerase chain reaction (qPCR), e.g., methylationsensitive restriction enzyme quantitative polymerase chain reaction(MSRE-qPCR)). In some instances, e.g., as set forth herein, a DMR thatis a methylation biomarker for advanced adenoma may also be useful inidentification of colorectal cancer.

DNA region: As used herein, “DNA region” refers to any contiguousportion of a larger DNA molecule. Those of skill in the art will befamiliar with techniques for determining whether a first DNA region anda second DNA region correspond, based, e.g., on sequence similarity(e.g, sequence identity or homology) of the first and second DNA regionsand/or context (e.g., the sequence identity or homology of nucleic acidsupstream and/or downstream of the first and second DNA regions).

Except as otherwise specified herein, sequences found in or relating tohumans (e.g., that hybridize to human DNA) are found in, based on,and/or derived from the example representative human genome sequencecommonly referred to, and known to those of skill in the art, as Homosapiens (human) genome assembly GRCh38, hg38, and/or Genome ReferenceConsortium Human Build 38. Those of skill in the art will furtherappreciate that DNA regions of hg38 can be referred to by a known systemincluding identification of particular nucleotide positions or rangesthereof in accordance with assigned numbering.

Dosing regimen: As used herein, the term “dosing regimen” can refer to aset of one or more same or different unit doses administered to asubject, typically including a plurality of unit doses administration ofeach of which is separated from administration of the others by a periodof time. In various embodiments, e.g., as set forth herein, one or moreor all unit doses of a dosing regimen may be the same or can vary (e.g.,increase over time, decrease over time, or be adjusted in accordancewith the subject and/or with a medical practitioner's determination). Invarious embodiments, e.g., as set forth herein, one or more or all ofthe periods of time between each dose may be the same or can vary (e.g.,increase over time, decrease over time, or be adjusted in accordancewith the subject and/or with a medical practitioner's determination). Insome embodiments, e.g., as set forth herein, a given therapeutic agenthas a recommended dosing regimen, which can involve one or more doses.Typically, at least one recommended dosing regimen of a marketed drug isknown to those of skill in the art. In some embodiments, e.g., as setforth herein, a dosing regimen is correlated with a desired orbeneficial outcome when administered across a relevant population (i.e.,is a therapeutic dosing regimen).

Downstream: As used herein, the term “downstream” means that a first DNAregion is closer, relative to a second DNA region, to the C-terminus ofa nucleic acid that includes the first DNA region and the second DNAregion.

Gene: As used herein, the term “gene” refers to a single DNA region,e.g., in a chromosome, that includes a coding sequence that encodes aproduct (e.g., an RNA product and/or a polypeptide product), togetherwith all, some, or none of the DNA sequences that contribute toregulation of the expression of coding sequence. In some embodiments,e.g., as set forth herein, a gene includes one or more non-codingsequences. In some particular embodiments, e.g., as set forth herein, agene includes exonic and intronic sequences. In some embodiments, e.g.,as set forth herein, a gene includes one or more regulatory elementsthat, for example, can control or impact one or more aspects of geneexpression (e.g., cell-type-specific expression, inducible expression,etc.). In some embodiments, e.g., as set forth herein, a gene includes apromoter. In some embodiments, e.g., as set forth herein, a geneincludes one or both of a (i) DNA nucleotides extending a predeterminednumber of nucleotides upstream of the coding sequence and (ii) DNAnucleotides extending a predetermined number of nucleotides downstreamof the coding sequence. In various embodiments, e.g., as set forthherein, the predetermined number of nucleotides can be 500 bp, 1 kb, 2kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 75 kb, or 100kb.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g., between nucleic acidmolecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Those of skill in the art will appreciate thathomology can be defined, e.g., by a percent identity or by a percenthomology (sequence similarity). In some embodiments, e.g., as set forthherein, polymeric molecules are considered to be “homologous” to oneanother if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In someembodiments, e.g., as set forth herein, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% similar.

Hybridize: As used herein, “hybridize” refers to the association of afirst nucleic acid with a second nucleic acid to form a double-strandedstructure, which association occurs through complementary pairing ofnucleotides. Those of skill in the art will recognize that complementarysequences, among others, can hybridize. In various embodiments, e.g., asset forth herein, hybridization can occur, for example, betweennucleotide sequences having at least 70% complementarity, e.g., at least75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%complementarity. Those of skill in the art will further appreciate thatwhether hybridization of a first nucleic acid and a second nucleic aciddoes or does not occur can dependence upon various reaction conditions.Conditions under which hybridization can occur are known in the art.

Hypomethylation: As used herein, the term “hypomethylation” refers tothe state of a methylation locus having at least one fewer methylatednucleotides in a state of interest as compared to a reference state(e.g., at least one fewer methylated nucleotides in colorectal cancerthan in healthy control).

Hypermethylation: As used herein, the term “hypermethylation” refers tothe state of a methylation locus having at least one more methylatednucleotide in a state of interest as compared to a reference state(e.g., at least one more methylated nucleotide in colorectal cancer thanin healthy control).

Identity, identical: As used herein, the terms “identity” and“identical” refers to the overall relatedness between polymericmolecules, e.g., between nucleic acid molecules (e.g., DNA moleculesand/or RNA molecules) and/or between polypeptide molecules. Methods forthe calculation of a percent identity as between two provided sequencesare known in the art. Calculation of the percent identity of two nucleicacid or polypeptide sequences, for example, can be performed by aligningthe two sequences (or the complement of one or both sequences) foroptimal comparison purposes (e.g., gaps can be introduced in one or bothof a first and a second sequences for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Thenucleotides or amino acids at corresponding positions are then compared.When a position in the first sequence is occupied by the same residue(e.g., nucleotide or amino acid) as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences and, optionally, takinginto account the number of gaps and the length of each gap, which mayneed to be introduced for optimal alignment of the two sequences. Thecomparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a computational algorithm, suchas BLAST (basic local alignment search tool).

“Improved,” “increased,” or “reduced”: As used herein, these terms, orgrammatically comparable comparative terms, indicate values that arerelative to a comparable reference measurement. For example, in someembodiments, e.g., as set forth herein, an assessed value achieved withan agent of interest may be “improved” relative to that obtained with acomparable reference agent or with no agent. Alternatively oradditionally, in some embodiments, e.g., as set forth herein, anassessed value in a subject or system of interest may be “improved”relative to that obtained in the same subject or system under differentconditions or at a different point in time (e.g., prior to or after anevent such as administration of an agent of interest), or in adifferent, comparable subject (e.g., in a comparable subject or systemthat differs from the subject or system of interest in presence of oneor more indicators of a particular disease, disorder or condition ofinterest, or in prior exposure to a condition or agent, etc.). In someembodiments, e.g., as set forth herein, comparative terms refer tostatistically relevant differences (e.g., differences of a prevalenceand/or magnitude sufficient to achieve statistical relevance). Those ofskill in the art will be aware, or will readily be able to determine, ina given context, a degree and/or prevalence of difference that isrequired or sufficient to achieve such statistical significance.

Methylation: As used herein, the term “methylation” includes methylationat any of (i) C5 position of cytosine; (ii) N4 position of cytosine; and(iii) the N6 position of adenine. Methylation also includes (iv) othertypes of nucleotide methylation. A nucleotide that is methylated can bereferred to as a “methylated nucleotide” or “methylated nucleotidebase.” In certain embodiments, e.g., as set forth herein, methylationspecifically refers to methylation of cytosine residues. In someinstances, methylation specifically refers to methylation of cytosineresidues present in CpG sites.

Methylation assay: As used herein, the term “methylation assay” refersto any technique that can be used to determine the methylation status ofa methylation locus.

Methylation biomarker: As used herein, the term “methylation biomarker”refers to a biomarker that is or includes at least one methylation locusand/or the methylation status of at least one methylation locus, e.g., ahypermethylated locus. In particular, a methylation biomarker is abiomarker characterized by a change between a first state and a secondstate (e.g., between a cancerous state and a non-cancerous state) inmethylation status of one or more nucleic acid loci.

Methylation locus: As used herein, the term “methylation locus” refersto a DNA region that includes at least one differentially methylatedregion. A methylation locus that includes a greater number or frequencyof methylated sites under a selected condition of interest, such as acancerous state, can be referred to as a hypermethylated locus. Amethylation locus that includes a smaller number or frequency ofmethylated sites under a selected condition of interest, such as acancerous state, can be referred to as a hypomethylated locus. In someinstances, e.g., as set forth herein, a methylation locus has a lengthof at least 10, at least 15, at least 20, at least 24, at least 50, orat least 75 base pairs. In some instances, e.g., as set forth herein, amethylation locus has a length of less than 1000, less than 750, lessthan 500, less than 350, less than 300, or less than 250 base pairs(e.g., where methylation status is determined using quantitativepolymerase chain reaction (qPCR), e.g., methylation sensitiverestriction enzyme quantitative polymerase chain reaction (MSRE-qPCR)).

Methylation site: As used herein, a methylation site refers to anucleotide or nucleotide position that is methylated in at least onecondition. In its methylated state, a methylation site can be referredto as a methylated site.

Methylation status: As used herein, “methylation status,” “methylationstate,” or “methylation profile” refer to the number, frequency, orpattern of methylation at methylation sites within a methylation locus.Accordingly, a change in methylation status between a first state and asecond state can be or include an increase in the number, frequency, orpattern of methylated sites, or can be or include a decrease in thenumber, frequency, or pattern of methylated sites. In various instances,a change in methylation status in a change in methylation value.

Methylation value: As used herein, the term “methylation value” refersto a numerical representation of a methylation status, e.g., in the formof number that represents the frequency or ratio of methylation of amethylation locus. In some instances, e.g., as set forth herein, amethylation value can be generated by a method that includes quantifyingthe amount of intact nucleic acid present in a sample followingrestriction digestion of the sample with a methylation dependentrestriction enzyme. In some instances, e.g., as set forth herein, amethylation value can be generated by a method that includes comparingamplification profiles after bisulfite reaction of a sample. In someinstances, e.g., as set forth herein, a methylation value can begenerated by comparing sequences of bisulfite-treated and untreatednucleic acids. In some instances, e.g., as set forth herein, amethylation value is, includes, or is based on a quantitative PCRresult.

Nucleic acid: As used herein, in its broadest sense, the term “nucleicacid” refers to any compound and/or substance that is or can beincorporated into an oligonucleotide chain. In some embodiments e.g., asset forth herein, a nucleic acid is a compound and/or substance that isor can be incorporated into an oligonucleotide chain via aphosphodiester linkage. As will be clear from context, in someembodiments e.g., as set forth herein, the term nucleic acid refers toan individual nucleic acid residue (e.g., a nucleotide and/ornucleoside), and in some embodiments e.g., as set forth herein refers toan polynucleotide chain comprising a plurality of individual nucleicacid residues. A nucleic acid can be or include DNA, RNA, or acombinations thereof. A nucleic acid can include natural nucleic acidresidues, nucleic acid analogs, and/or synthetic residues. In someembodiments e.g., as set forth herein, a nucleic acid includes naturalnucleotides (e.g., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). Insome embodiments e.g., as set forth herein, a nucleic acid is orincludes of one or more nucleotide analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguano sine, 8-oxoadenosine,8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases,intercalated bases, and combinations thereof).

In some embodiments e.g., as set forth herein, a nucleic acid has anucleotide sequence that encodes a functional gene product such as anRNA or protein. In some embodiments e.g., as set forth herein, a nucleicacid includes one or more introns. In some embodiments e.g., as setforth herein, a nucleic acid includes one or more genes. In someembodiments e.g., as set forth herein, nucleic acids are prepared by oneor more of isolation from a natural source, enzymatic synthesis bypolymerization based on a complementary template (in vivo or in vitro),reproduction in a recombinant cell or system, and chemical synthesis.

In some embodiments e.g., as set forth herein, a nucleic acid analogdiffers from a nucleic acid in that it does not utilize a phosphodiesterbackbone. For example, in some embodiments e.g., as set forth herein, anucleic acid can include one or more peptide nucleic acids, which areknown in the art and have peptide bonds instead of phosphodiester bondsin the backbone. Alternatively or additionally, in some embodimentse.g., as set forth herein, a nucleic acid has one or morephosphorothioate and/or 5′-N-phosphoramidite linkages rather thanphosphodiester bonds. In some embodiments e.g., as set forth herein, anucleic acid comprises one or more modified sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose) ascompared with those in natural nucleic acids.

In some embodiments, e.g., as set forth herein, a nucleic acid is orincludes at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150,160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000 or more residues. In some embodiments, e.g., as setforth herein, a nucleic acid is partly or wholly single stranded, orpartly or wholly double stranded.

Nucleic acid detection assay: As used herein, the term “nucleic aciddetection assay” refers to any method of determining the nucleotidecomposition of a nucleic acid of interest. Nucleic acid detection assaysinclude but are not limited to, DNA sequencing methods, polymerase chainreaction-based methods, probe hybridization methods, ligase chainreaction, etc.

Nucleotide: As used herein, the term “nucleotide” refers to a structuralcomponent, or building block, of polynucleotides, e.g., of DNA and/orRNA polymers. A nucleotide includes of a base (e.g., adenine, thymine,uracil, guanine, or cytosine) and a molecule of sugar and at least onephosphate group. As used herein, a nucleotide can be a methylatednucleotide or an un-methylated nucleotide. Those of skill in the artwill appreciate that nucleic acid terminology, such as, as examples,“locus” or “nucleotide” can refer to both a locus or nucleotide of asingle nucleic acid molecule and/or to the cumulative population of locior nucleotides within a plurality of nucleic acids (e.g., a plurality ofnucleic acids in a sample and/or representative of a subject) that arerepresentative of the locus or nucleotide (e.g., having the sameidentical nucleic acid sequence and/or nucleic acid sequence context, orhaving a substantially identical nucleic acid sequence and/or nucleicacid context).

Oligonucleotide primer: As used herein, the term oligonucleotide primer,or primer, refers to a nucleic acid molecule used, capable of beingused, or for use in, generating amplicons from a template nucleic acidmolecule. Under transcription-permissive conditions (e.g., in thepresence of nucleotides and a DNA polymerase, and at a suitabletemperature and pH), an oligonucleotide primer can provide a point ofinitiation of transcription from a template to which the oligonucleotideprimer hybridizes. Typically, an oligonucleotide primer is asingle-stranded nucleic acid between 5 and 200 nucleotides in length.Those of skill in the art will appreciate that optimal primer length forgenerating amplicons from a template nucleic acid molecule can vary withconditions including temperature parameters, primer composition, andtranscription or amplification method. A pair of oligonucleotideprimers, as used herein, refers to a set of two oligonucleotide primersthat are respectively complementary to a first strand and a secondstrand of a template double-stranded nucleic acid molecule. First andsecond members of a pair of oligonucleotide primers may be referred toas a “forward” oligonucleotide primer and a “reverse” oligonucleotideprimer, respectively, with respect to a template nucleic acid strand, inthat the forward oligonucleotide primer is capable of hybridizing with anucleic acid strand complementary to the template nucleic acid strand,the reverse oligonucleotide primer is capable of hybridizing with thetemplate nucleic acid strand, and the position of the forwardoligonucleotide primer with respect to the template nucleic acid strandis 5′ of the position of the reverse oligonucleotide primer sequencewith respect to the template nucleic acid strand. It will be understoodby those of skill in the art that the identification of a first andsecond oligonucleotide primer as forward and reverse oligonucleotideprimers, respectively, is arbitrary inasmuch as these identifiers dependupon whether a given nucleic acid strand or its complement is utilizedas a template nucleic acid molecule.

Overlapping: The term “overlapping” is used herein in reference to tworegions of DNA, each of which contains a sub-sequence that issubstantially identical to a sub-sequence of the same length in theother region (e.g., the two regions of DNA have a common sub-sequence).“Substantially identical” means that the two identically-longsub-sequences differ by fewer than a given number of base pairs. Incertain instances, e.g., as set forth herein, each sub-sequence has alength of at least 20 base pairs that differ by fewer than 4, 3, 2, or 1base pairs from each other (e.g., the two sub-sequences having at least80%, at least 85%, at least 90%, at least 95% similarity, at least 97%similarity, at least 98% similarity, at least 99% similarity, or atleast 99.5% similarity). In certain instances, e.g., as set forthherein, each sub-sequence has a length of at least 24 base pairs thatdiffer by fewer than 5, 4, 3, 2, or 1 base pairs (e.g., the twosub-sequences having at least 80%, at least 85%, at least 90%, at least95% similarity, at least 97% similarity, at least 98% similarity, atleast 99% similarity, or at least 99.5% similarity). In certaininstances, e.g., as set forth herein, each sub-sequence has a length ofat least 50 base pairs that differ by fewer than 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 base pairs (e.g., the two sub-sequences having at least 80%,at least 85%, at least 90%, at least 95% similarity, at least 97%similarity, at least 98% similarity, at least 99% similarity, or atleast 99.5% similarity). In certain instances, e.g., as set forthherein, each sub-sequence has a length of at least 100 base pairs thatdiffer by fewer than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base pairs(e.g., the two sub-sequences having at least 80%, at least 85%, at least90%, at least 95% similarity, at least 97% similarity, at least 98%similarity, at least 99% similarity, or at least 99.5% similarity). Incertain instances, e.g., as set forth herein, each sub-sequence has alength of at least 200 base pairs that differ by fewer than 40, 30, 20,15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base pairs (e.g., the twosub-sequences having at least 80%, at least 85%, at least 90%, at least95% similarity, at least 97% similarity, at least 98% similarity, atleast 99% similarity, or at least 99.5% similarity). In certaininstances, e.g., as set forth herein, each sub-sequence has a length ofat least 250 base pairs that differ by fewer than 50, 40, 30, 20, 15,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base pairs (e.g., the two sub-sequenceshaving at least 80%, at least 85%, at least 90%, at least 95%similarity, at least 97% similarity, at least 98% similarity, at least99% similarity, or at least 99.5% similarity). In certain instances,e.g., as set forth herein, each sub-sequence has a length of at least300 base pairs that differ by fewer than 60, 50, 40, 30, 20, 15, 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 base pairs (e.g., the two sub-sequences havingat least 80%, at least 85%, at least 90%, at least 95% similarity, atleast 97% similarity, at least 98% similarity, at least 99% similarity,or at least 99.5% similarity). In certain instances, e.g., as set forthherein, each sub-sequence has a length of at least 500 base pairs thatdiffer by fewer than 100, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 base pairs (e.g., the two sub-sequences having at least 80%,at least 85%, at least 90%, at least 95% similarity, at least 97%similarity, at least 98% similarity, at least 99% similarity, or atleast 99.5% similarity). In certain instances, e.g., as set forthherein, each sub-sequence has a length of at least 1000 base pairs thatdiffer by fewer than 200, 100, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 base pairs (e.g., the two sub-sequences having at least80%, at least 85%, at least 90%, at least 95% similarity, at least 97%similarity, at least 98% similarity, at least 99% similarity, or atleast 99.5% similarity). In certain instances, e.g., as set forthherein, the subsequence of a first region of the two regions of DNA maycomprise the entirety of the second region of the two regions of DNA (orvice versa) (e.g., the common sub-sequence may contain the whole ofeither or both regions).

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to a composition in which an active agent isformulated together with one or more pharmaceutically acceptablecarriers. In some embodiments, e.g., as set forth herein, the activeagent is present in a unit dose amount appropriate for administration toa subject, e.g., in a therapeutic regimen that shows a statisticallysignificant probability of achieving a predetermined therapeutic effectwhen administered to a relevant population. In some embodiments, e.g.,as set forth herein, a pharmaceutical composition can be formulated foradministration in a particular form (e.g., in a solid form or a liquidform), and/or can be specifically adapted for, for example: oraladministration (for example, as a drenche (aqueous or non-aqueoussolutions or suspensions), tablet, capsule, bolus, powder, granule,paste, etc., which can be formulated specifically for example forbuccal, sublingual, or systemic absorption); parenteral administration(for example, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation, etc.); topical application (for example,as a cream, ointment, patch or spray applied for example to skin, lungs,or oral cavity); intravaginal or intrarectal administration (forexample, as a pessary, suppository, cream, or foam); ocularadministration; nasal or pulmonary administration, etc.

Pharmaceutically acceptable: As used herein, the term “pharmaceuticallyacceptable,” as applied to one or more, or all, component(s) forformulation of a composition as disclosed herein, means that eachcomponent must be compatible with the other ingredients of thecomposition and not deleterious to the recipient thereof.

Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” refers to apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, that facilitates formulation and/or modifies bioavailabilityof an agent, e.g., a pharmaceutical agent. Some examples of materialswhich can serve as pharmaceutically-acceptable carriers include: sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients, such as cocoa butter andsuppository waxes; oils, such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil; glycols, such aspropylene glycol; polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides; and other non-toxic compatiblesubstances employed in pharmaceutical formulations.

Prevent or prevention: The terms “prevent” and “prevention,” as usedherein in connection with the occurrence of a disease, disorder, orcondition, refers to reducing the risk of developing the disease,disorder, or condition; delaying onset of the disease, disorder, orcondition; delaying onset of one or more characteristics or symptoms ofthe disease, disorder, or condition; and/or to reducing the frequencyand/or severity of one or more characteristics or symptoms of thedisease, disorder, or condition. Prevention can refer to prevention in aparticular subject or to a statistical impact on a population ofsubjects. Prevention can be considered complete when onset of a disease,disorder, or condition has been delayed for a predefined period of time.

Probe: As used herein, the term “probe” refers to a single- ordouble-stranded nucleic acid molecule that is capable of hybridizingwith a complementary target and includes a detectable moiety. In certainembodiments, e.g., as set forth herein, a probe is a restriction digestproduct or is a synthetically produced nucleic acid, e.g., a nucleicacid produced by recombination or amplification. In some instances,e.g., as set forth herein, a probe is a capture probe useful indetection, identification, and/or isolation of a target sequence, suchas a gene sequence. In various instances, e.g., as set forth herein, adetectable moiety of probe can be, e.g., an enzyme (e.g., ELISA, as wellas enzyme-based histochemical assays), fluorescent moiety, radioactivemoiety, or moiety associated with a luminescence signal.

Prognosis: As used herein, the term “prognosis” refers to determiningthe qualitative of quantitative probability of at least one possiblefuture outcome or event. As used herein, a prognosis can be adetermination of the likely course of a disease, disorder, or conditionsuch as cancer in a subject, a determination regarding the lifeexpectancy of a subject, or a determination regarding response totherapy, e.g., to a particular therapy.

Prognostic information: As used herein, the term “prognosticinformation” refers to information useful in providing a prognosis.Prognostic information can include, without limitation, biomarker statusinformation.

Promoter: As used herein, a “promoter” can refer to a DNA regulatoryregion that directly or indirectly (e.g., through promoter-boundproteins or substances) associates with an RNA polymerase andparticipates in initiation of transcription of a coding sequence.

Reference: As used herein describes a standard or control relative towhich a comparison is performed. For example, in some embodiments, e.g.,as set forth herein, an agent, subject, animal, individual, population,sample, sequence, or value of interest is compared with a reference orcontrol agent, subject, animal, individual, population, sample,sequence, or value. In some embodiments, e.g., as set forth herein, areference or characteristic thereof is tested and/or determinedsubstantially simultaneously with the testing or determination of thecharacteristic in a sample of interest. In some embodiments, e.g., asset forth herein, a reference is a historical reference, optionallyembodied in a tangible medium. Typically, as would be understood bythose of skill in the art, a reference is determined or characterizedunder comparable conditions or circumstances to those under assessment,e.g., with regard to a sample. Those skilled in the art will appreciatewhen sufficient similarities are present to justify reliance on and/orcomparison to a particular possible reference or control.

Risk: As used herein with respect to a disease, disorder, or condition,the term “risk” refers to the qualitative of quantitative probability(whether expressed as a percentage or otherwise) that a particularindividual will develop the disease, disorder, or condition. In someembodiments, e.g., as set forth herein, risk is expressed as apercentage. In some embodiments, e.g., as set forth herein, a risk is aqualitative of quantitative probability that is equal to or greater than0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or100%. In some embodiments, e.g., as set forth herein, risk is expressedas a qualitative of quantitative level of risk relative to a referencerisk or level or the risk of the same outcome attributed to a reference.In some embodiments, e.g., as set forth herein, relative risk isincreased or decreased in comparison to the reference sample by a factorof 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9,10, or more.

Sample: As used herein, the term “sample” typically refers to an aliquotof material obtained or derived from a source of interest. In someembodiments, e.g., as set forth herein, a source of interest is abiological or environmental source. In some embodiments, e.g., as setforth herein, a sample is a “primary sample” obtained directly from asource of interest. In some embodiments, e.g., as set forth herein, aswill be clear from context, the term “sample” refers to a preparationthat is obtained by processing of a primary sample (e.g., by removingone or more components of and/or by adding one or more agents to aprimary sample). Such a “processed sample” can include, for examplecells, nucleic acids, or proteins extracted from a sample or obtained bysubjecting a primary sample to techniques such as amplification orreverse transcription of nucleic acids, isolation and/or purification ofcertain components, etc.

In certain instances, e.g., as set forth herein, a processed sample canbe a DNA sample that has been amplified (e.g., pre-amplified). Thus, invarious instances, e.g., as set forth herein, an identified sample canrefer to a primary form of the sample or to a processed form of thesample. In some instances, e.g., as set forth herein, a sample that isenzyme-digested DNA can refer to primary enzyme-digested DNA (theimmediate product of enzyme digestion) or a further processed samplesuch as enzyme-digested DNA that has been subject to an amplificationstep (e.g., an intermediate amplification step, e.g., pre-amplification)and/or to a filtering step, purification step, or step that modifies thesample to facilitate a further step, e.g., in a process of determiningmethylation status (e.g., methylation status of a primary sample of DNAand/or of DNA as it existed in its original source context).

Screening: As used herein, the term “screening” refers to any method,technique, process, or undertaking intended to generate diagnosticinformation and/or prognostic information. Accordingly, those of skillin the art will appreciate that the term screening encompasses method,technique, process, or undertaking that determines whether an individualhas, is likely to have or develop, or is at risk of having or developinga disease, disorder, or condition, e.g., colorectal cancer.

Specificity: As used herein, the “specificity” of a biomarker refers tothe percentage of samples that are characterized by absence of the eventor state of interest for which measurement of the biomarker accuratelyindicates absence of the event or state of interest (true negativerate). In various embodiments, e.g., as set forth herein,characterization of the negative samples is independent of thebiomarker, and can be achieved by any relevant measure, e.g., anyrelevant measure known to those of skill in the art. Thus, specificityreflects the probability that the biomarker would detect the absence ofthe event or state of interest when measured in a sample notcharacterized that event or state of interest. In particular embodimentsin which the event or state of interest is colorectal cancer, e.g., asset forth herein, specificity refers to the probability that a biomarkerwould detect the absence of colorectal cancer in a subject lackingcolorectal cancer. Lack of colorectal cancer can be determined, e.g., byhistology.

Sensitivity: As used herein, the “sensitivity” of a biomarker refers tothe percentage of samples that are characterized by the presence of theevent or state of interest for which measurement of the biomarkeraccurately indicates presence of the event or state of interest (truepositive rate). In various embodiments, e.g., as set forth herein,characterization of the positive samples is independent of thebiomarker, and can be achieved by any relevant measure, e.g., anyrelevant measure known to those of skill in the art. Thus, sensitivityreflects the probability that a biomarker would detect the presence ofthe event or state of interest when measured in a sample characterizedby presence of that event or state of interest. In particularembodiments in which the event or state of interest is colorectalcancer, e.g., as set forth herein, sensitivity refers to the probabilitythat a biomarker would detect the presence of colorectal cancer in asubject that has colorectal cancer. Presence of colorectal cancer can bedetermined, e.g., by histology.

Solid Tumor: As used herein, the term “solid tumor” refers to anabnormal mass of tissue including cancer cells. In various embodiments,e.g., as set forth herein, a solid tumor is or includes an abnormal massof tissue that does not contain cysts or liquid areas. In someembodiments, e.g., as set forth herein, a solid tumor can be benign; insome embodiments, a solid tumor can be malignant. Examples of solidtumors include carcinomas, lymphomas, and sarcomas. In some embodiments,e.g., as set forth herein, solid tumors can be or include adrenal, bileduct, bladder, bone, brain, breast, cervix, colon, endometrium,esophagum, eye, gall bladder, gastrointestinal tract, kidney, larynx,liver, lung, nasal cavity, nasopharynx, oral cavity, ovary, penis,pituitary, prostate, retina, salivary gland, skin, small intestine,stomach, testis, thymus, thyroid, uterine, vaginal, and/or vulvaltumors.

Stage of cancer: As used herein, the term “stage of cancer” refers to aqualitative or quantitative assessment of the level of advancement of acancer. In some embodiments, e.g., as set forth herein, criteria used todetermine the stage of a cancer can include, but are not limited to, oneor more of where the cancer is located in a body, tumor size, whetherthe cancer has spread to lymph nodes, whether the cancer has spread toone or more different parts of the body, etc. In some embodiments, e.g.,as set forth herein, cancer can be staged using the so-called TNMSystem, according to which T refers to the size and extent of the maintumor, usually called the primary tumor; N refers to the number ofnearby lymph nodes that have cancer; and M refers to whether the cancerhas metastasized. In some embodiments, e.g., as set forth herein, acancer can be referred to as Stage 0 (abnormal cells are present buthave not spread to nearby tissue, also called carcinoma in situ, or CIS;CIS is not cancer, but it can become cancer), Stage I-III (cancer ispresent; the higher the number, the larger the tumor and the more it hasspread into nearby tissues), or Stage IV (the cancer has spread todistant parts of the body). In some embodiments, e.g., as set forthherein, a cancer can be assigned to a stage selected from the groupconsisting of: in situ (abnormal cells are present but have not spreadto nearby tissue); localized (cancer is limited to the place where itstarted, with no sign that it has spread); regional (cancer has spreadto nearby lymph nodes, tissues, or organs): distant (cancer has spreadto distant parts of the body); and unknown (there is not enoughinformation to identify cancer stage).

Susceptible to: An individual who is “susceptible to” a disease,disorder, or condition is at risk for developing the disease, disorder,or condition. In some embodiments, e.g., as set forth herein, anindividual who is susceptible to a disease, disorder, or condition doesnot display any symptoms of the disease, disorder, or condition. In someembodiments, e.g., as set forth herein, an individual who is susceptibleto a disease, disorder, or condition has not been diagnosed with thedisease, disorder, and/or condition. In some embodiments, e.g., as setforth herein, an individual who is susceptible to a disease, disorder,or condition is an individual who has been exposed to conditionsassociated with, or presents a biomarker status (e.g., a methylationstatus) associated with, development of the disease, disorder, orcondition. In some embodiments, e.g., as set forth herein, a risk ofdeveloping a disease, disorder, and/or condition is a population-basedrisk (e.g., family members of individuals suffering from the disease,disorder, or condition).

Subject: As used herein, the term “subject” refers to an organism,typically a mammal (e.g., a human). In some embodiments, e.g., as setforth herein, a subject is suffering from a disease, disorder orcondition. In some embodiments, e.g., as set forth herein, a subject issusceptible to a disease, disorder, or condition. In some embodiments,e.g., as set forth herein, a subject displays one or more symptoms orcharacteristics of a disease, disorder or condition. In someembodiments, e.g., as set forth herein, a subject is not suffering froma disease, disorder or condition. In some embodiments, e.g., as setforth herein, a subject does not display any symptom or characteristicof a disease, disorder, or condition. In some embodiments, e.g., as setforth herein, a subject is someone with one or more featurescharacteristic of susceptibility to or risk of a disease, disorder, orcondition. In some embodiments, e.g., as set forth herein, a subject isa patient. In some embodiments, e.g., as set forth herein, a subject isan individual to whom diagnosis has been performed and/or to whomtherapy has been administered. In some instances, e.g., as set forthherein, a human subject can be interchangeably referred to as an“individual.”

Therapeutic agent: As used herein, the term “therapeutic agent” refersto any agent that elicits a desired pharmacological effect whenadministered to a subject. In some embodiments, e.g., as set forthherein, an agent is considered to be a therapeutic agent if itdemonstrates a statistically significant effect across an appropriatepopulation. In some embodiments, e.g., as set forth herein, theappropriate population can be a population of model organisms or a humanpopulation. In some embodiments, e.g., as set forth herein, anappropriate population can be defined by various criteria, such as acertain age group, gender, genetic background, preexisting clinicalconditions, etc. In some embodiments, e.g., as set forth herein, atherapeutic agent is a substance that can be used for treatment of adisease, disorder, or condition. In some embodiments, e.g., as set forthherein, a therapeutic agent is an agent that has been or is required tobe approved by a government agency before it can be marketed foradministration to humans. In some embodiments, e.g., as set forthherein, a therapeutic agent is an agent for which a medical prescriptionis required for administration to humans.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount that produces adesired effect for which it is administered. In some embodiments, e.g.,as set forth herein, the term refers to an amount that is sufficient,when administered to a population suffering from or susceptible to adisease, disorder, or condition, in accordance with a therapeutic dosingregimen, to treat the disease, disorder, or condition. Those of ordinaryskill in the art will appreciate that the term therapeutically effectiveamount does not in fact require successful treatment be achieved in aparticular individual. Rather, a therapeutically effective amount can bean amount that provides a particular desired pharmacological response ina significant number of subjects when administered to individuals inneed of such treatment. In some embodiments, e.g., as set forth herein,reference to a therapeutically effective amount can be a reference to anamount as measured in one or more specific tissues (e.g., a tissueaffected by the disease, disorder or condition) or fluids (e.g., blood,saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill inthe art will appreciate that, in some embodiments, a therapeuticallyeffective amount of a particular agent can be formulated and/oradministered in a single dose. In some embodiments, e.g., as set forthherein, a therapeutically effective agent can be formulated and/oradministered in a plurality of doses, for example, as part of amulti-dose dosing regimen.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to administration of a therapy that partially orcompletely alleviates, ameliorates, relieves, inhibits, delays onset of,reduces severity of, and/or reduces incidence of one or more symptoms,features, and/or causes of a particular disease, disorder, or condition,or is administered for the purpose of achieving any such result. In someembodiments, e.g., as set forth herein, such treatment can be of asubject who does not exhibit signs of the relevant disease, disorder, orcondition and/or of a subject who exhibits only early signs of thedisease, disorder, or condition. Alternatively or additionally, suchtreatment can be of a subject who exhibits one or more established signsof the relevant disease, disorder and/or condition. In some embodiments,e.g., as set forth herein, treatment can be of a subject who has beendiagnosed as suffering from the relevant disease, disorder, and/orcondition. In some embodiments, e.g., as set forth herein, treatment canbe of a subject known to have one or more susceptibility factors thatare statistically correlated with increased risk of development of therelevant disease, disorder, or condition. In various examples, treatmentis of a cancer.

Upstream: As used herein, the term “upstream” means a first DNA regionis closer, relative to a second DNA region, to the N-terminus of anucleic acid that includes the first DNA region and the second DNAregion.

Unit dose: As used herein, the term “unit dose” refers to an amountadministered as a single dose and/or in a physically discrete unit of apharmaceutical composition. In many embodiments, e.g., as set forthherein, a unit dose contains a predetermined quantity of an activeagent. In some embodiments, e.g., as set forth herein, a unit dosecontains an entire single dose of the agent. In some embodiments, e.g.,as set forth herein, more than one unit dose is administered to achievea total single dose. In some embodiments, e.g., as set forth herein,administration of multiple unit doses is required, or expected to berequired, in order to achieve an intended effect. A unit dose can be,for example, a volume of liquid (e.g., an acceptable carrier) containinga predetermined quantity of one or more therapeutic moieties, apredetermined amount of one or more therapeutic moieties in solid form,a sustained release formulation or drug delivery device containing apredetermined amount of one or more therapeutic moieties, etc. It willbe appreciated that a unit dose can be present in a formulation thatincludes any of a variety of components in addition to the therapeuticagent(s). For example, acceptable carriers (e.g., pharmaceuticallyacceptable carriers), diluents, stabilizers, buffers, preservatives,etc., can be included. It will be appreciated by those skilled in theart, in many embodiments, e.g., as set forth herein, a total appropriatedaily dosage of a particular therapeutic agent can comprise a portion,or a plurality, of unit doses, and can be decided, for example, by amedical practitioner within the scope of sound medical judgment. In someembodiments, e.g., as set forth herein, the specific effective doselevel for any particular subject or organism can depend upon a varietyof factors including the disorder being treated and the severity of thedisorder; activity of specific active compound employed; specificcomposition employed; age, body weight, general health, sex and diet ofthe subject; time of administration, and rate of excretion of thespecific active compound employed; duration of the treatment; drugsand/or additional therapies used in combination or coincidental withspecific compound(s) employed, and like factors well known in themedical arts

Unmethylated: As used herein, the terms “unmethylated” and“non-methylated” are used interchangeable and mean that an identifiedDNA region includes no methylated nucleotides.

Variant: As used herein, the term “variant” refers to an entity thatshows significant structural identity with a reference entity butdiffers structurally from the reference entity in the presence, absence,or level of one or more chemical moieties as compared with the referenceentity. In some embodiments, e.g., as set forth herein, a variant alsodiffers functionally from its reference entity. In general, whether aparticular entity is properly considered to be a “variant” of areference entity is based on its degree of structural identity with thereference entity. A variant can be a molecule comparable, but notidentical to, a reference. For example, a variant nucleic acid candiffer from a reference nucleic acid at one or more differences innucleotide sequence. In some embodiments, e.g., as set forth herein, avariant nucleic acid shows an overall sequence identity with a referencenucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, or 99%. In many embodiments, e.g., as set forthherein, a nucleic acid of interest is considered to be a “variant” of areference nucleic acid if the nucleic acid of interest has a sequencethat is identical to that of the reference but for a small number ofsequence alterations at particular positions. In some embodiments, e.g.,as set forth herein, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substituted residues as compared with a reference. In some embodiments,e.g., as set forth herein, a variant has not more than 5, 4, 3, 2, or 1residue additions, substitutions, or deletions as compared with thereference. In various embodiments, e.g., as set forth herein, the numberof additions, substitutions, or deletions is fewer than about 25, about20, about 19, about 18, about 17, about 16, about 15, about 14, about13, about 10, about 9, about 8, about 7, about 6, and commonly are fewerthan about 5, about 4, about 3, or about 2 residues.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects, features, and advantages ofthe present disclosure will become more apparent and better understoodby referring to the following description taken in conjunction with theaccompanying drawings, in which:

FIG. 1 is a schematic showing an example MSRE-qPCR approach.

FIG. 2 is a table showing characteristics of a first subject group of 70human subjects. FIG. 2 provides the percent female, percent male, agerange, and BMI of subjects. FIG. 2 further distinguishes the types ofcolorectal cancer identified in the first subject group as localized oradvanced based on histological evaluation, and as a proximal or distalbased on colonoscopy evaluation of colon.

FIG. 3 is a table showing characteristics of a second subject group of63 human subjects. FIG. 3 provides the percent female, percent male, agerange, and BMI of subjects. FIG. 3 further distinguishes the types ofcolorectal cancer identified in the second subject group as localized oradvanced based on histological evaluation, and as a proximal or distalbased on colonoscopy evaluation of colon.

FIG. 4 includes panels A and B. Panel A of FIG. 4 is a graph showingperformance of colorectal cancer screening using a representativeproof-of-principle panel of DMRs on the second subject group. ROC curveand AUC for all subjects of the second subject group are shown. Panel Bof FIG. 4 is a chart showing accuracy values, including, from left toright, overall sensitivity of colorectal screening for colorectalcancer, sensitivity of colorectal screening for localized colorectalcancer, sensitivity of colorectal screening for advanced colorectalcancer, sensitivity of colorectal screening for proximal colorectalcancer, sensitivity of colorectal screening for distal colorectalcancer, and specificity of colorectal screening for control subjects(healthy subjects and subjects with non-advanced adenoma).

FIG. 5 is a graph representing Ct values from MSRE-qPCR of ALK '434 forsubjects with colorectal cancer and control subjects (healthy subjectsand subjects with non-advanced adenoma). Data represent the secondsubject group (63 subjects) used for testing. For display purposes, Ctvalues are subtracted from 45 (45−Ct). Higher 45−Ct values correspond tohigher methylation status, demonstrating hypermethylation in subjectswith colorectal cancer.

FIG. 6 is a graph representing Ct values from MSRE-qPCR of FGF14 '577DMR for subjects with colorectal cancer and control subjects (healthysubjects and subjects with non-advanced adenoma). Data represent thesecond subject group (63 subjects) used for testing. For displaypurposes, Ct values are subtracted from 45 (45−Ct). Higher 45−Ct valuescorrespond to higher methylation status, demonstrating hypermethylationin subjects with colorectal cancer.

FIG. 7 is a graph representing Ct values from MSRE-qPCR of PDGFD '388for subjects with colorectal cancer and control subjects (healthysubjects and subjects with non-advanced adenoma). Data represent thesecond subject group (63 subjects) used for testing. For displaypurposes, Ct values are subtracted from 45 (45−Ct). Higher 45−Ct valuescorrespond to higher methylation status, demonstrating hypermethylationin subjects with colorectal cancer.

FIG. 8 is a graph representing Ct values from MSRE-qPCR of JAM2 '320 forsubjects with colorectal cancer and control subjects (healthy subjectsand subjects with non-advanced adenoma). Data represent the d secondsubject group (63 subjects) used for testing. For display purposes, Ctvalues are subtracted from 45 (45−Ct). Higher 45−Ct values correspond tohigher methylation status, demonstrating hypermethylation in subjectswith colorectal cancer.

FIG. 9 is a graph representing Ct values from MSRE-qPCR of LONRF2 '281for subjects with colorectal cancer and control subjects (healthysubjects and subjects with non-advanced adenoma). Data represent thesecond subject group (63 subjects) used for testing. For displaypurposes, Ct values are subtracted from 45 (45−Ct). Higher 45−Ct valuescorrespond to higher methylation status, demonstrating hypermethylationin subjects with colorectal cancer.

FIG. 10 is a table showing characteristics of a third subject group of82 human subjects. FIG. 10 provides the percent female, percent male,age range, and BMI of subjects diagnosed by screening using 28colorectal cancer DMRs of the present disclosure. FIG. 10 furtherdistinguishes the types of colorectal cancer identified in the thirdsubject group as localized or advanced based on histological evaluation,and as a proximal or distal based on colonoscopy evaluation of colon.

FIG. 11 is a graph showing performance of colorectal cancer screeningusing a 28 DMR panel in the third subject group. ROC curve and AUC forall subjects of the third subject group are shown. ROC-curve analysisshowed that a 28 DMR panel achieved general colorectal cancersensitivity of 79%, with 75% sensitivity for localized (early) cancerand 84% sensitivity for advanced cancer, on a very stable specificity of87% at AUC 82% (see also Table 15).

FIG. 12 is a graph representing Ct values from MSRE-qPCR of ZNF471 '527for subjects with colorectal cancer and control subjects (healthysubjects and subjects with non-advanced adenoma). Data represent thethird subject group (82 subjects). For display purposes, Ct values aresubtracted from 45 (45−Ct). Higher 45−Ct values correspond to highermethylation status, demonstrating hypermethylation in subjects withcolorectal cancer.

FIG. 13 is a graph representing Ct values from MSRE-qPCR of FGF14 '577for subjects with colorectal cancer and control subjects (healthysubjects and subjects with non-advanced adenoma). Data represent thethird subject group (82 subjects). For display purposes, Ct values aresubtracted from 45 (45−Ct). Higher 45−Ct values correspond to highermethylation status, demonstrating hypermethylation in subjects withcolorectal cancer.

FIG. 14 is a graph representing Ct values from MSRE-qPCR of PDGFD '388for subjects with colorectal cancer and control subjects (healthysubjects and subjects with non-advanced adenoma). Data represent thethird subject group (82 subjects). For display purposes, Ct values aresubtracted from 45 (45−Ct). Higher 45−Ct values correspond to highermethylation status, demonstrating hypermethylation in subjects withcolorectal cancer.

FIG. 15 is a graph representing Ct values from MSRE-qPCR of ADAMTS2 '254for subjects with colorectal cancer and control subjects (healthysubjects and subjects with non-advanced adenoma). Data represent thethird subject group (82 subjects). For display purposes, Ct values aresubtracted from 45 (45−Ct). Higher 45−Ct values correspond to highermethylation status, demonstrating hypermethylation in subjects withcolorectal cancer.

FIG. 16 is a graph representing Ct values from MSRE-qPCR of ZNF471 '558(which DMR is alternatively referred to herein as ZNF471_2) for subjectswith colorectal cancer and control subjects (healthy subjects andsubjects with non-advanced adenoma). Data represent the third subjectgroup (82 subjects). For display purposes, Ct values are subtracted from45 (45−Ct). Higher 45−Ct values correspond to higher methylation status,demonstrating hypermethylation in subjects with colorectal cancer.

FIG. 17 is a graph representing Ct values from MSRE-qPCR of ST6GALNAC5'456 for subjects with colorectal cancer and control subjects (healthysubjects and subjects with non-advanced adenoma). Data represent thethird subject group (82 subjects). For display purposes, Ct values aresubtracted from 45 (45−Ct). Higher 45−Ct values correspond to highermethylation status, demonstrating hypermethylation in subjects withcolorectal cancer.

FIG. 18 is a graph representing Ct values from MSRE-qPCR of ZNF542 '525for subjects with colorectal cancer and control subjects (healthysubjects and subjects with non-advanced adenoma). Data represent thethird subject group (82 subjects). For display purposes, Ct values aresubtracted from 45 (45−Ct). Higher 45−Ct values correspond to highermethylation status, demonstrating hypermethylation in subjects withcolorectal cancer.

FIG. 19 is a graph representing Ct values from MSRE-qPCR of LONRF2 '281for subjects with colorectal cancer and control subjects (healthysubjects and subjects with non-advanced adenoma). Data represent thethird subject group (82 subjects). For display purposes, Ct values aresubtracted from 45 (45−Ct). Higher 45−Ct values correspond to highermethylation status, demonstrating hypermethylation in subjects withcolorectal cancer.

FIG. 20 is a schematic showing example methylation changes inmethylation status between normal and cancer cells, and furtherindicates how changes in methylation status can impact gene expressiondifferences between normal and cancer cells.

FIG. 21 is a table showing the characteristics of a subject group of 215subjects used as a training set. FIG. 21 provides the number of females,number of males, age range, and colorectal cancer status of subjects.FIG. 21 further distinguishes the types of colorectal cancer identifiedin those having colorectal cancer in the subject group as localized oradvanced based on histological evaluation, and as a proximal or distalbased on colonoscopy evaluation of colon.

FIG. 22 is a table showing the characteristics of a fourth subject groupof 774 subjects used as a validation set. FIG. 22 provides the number offemales, number of males, age range, and cancer status of subjects. FIG.22 further distinguishes the types of cancer identified in those havingcancer in the subject group as localized or advanced based onhistological evaluation.

FIG. 23 is a graph showing performance of a 3-marker panel forcolorectal cancer screening using DMRs of Table 18. ROC curve andperformance features for all subjects of the fourth, validation subjectgroup are shown.

FIG. 24 is a graph showing performance of a 5-marker panel forcolorectal cancer screening using DMRs of Table 19. ROC curve andperformance features for all subjects of the fourth, validation subjectgroup are shown.

FIG. 25 is a graph showing performance of a 6-marker panel forcolorectal cancer screening using DMRs of Table 20. ROC curve andperformance features for all subjects of the fourth, validation subjectgroup are shown.

FIG. 26 is a graph representing Ct values from MSRE-qPCR of ZNF132 '415for subjects with colorectal cancer and control subjects (healthysubjects, subjects with non-advanced adenoma, and subjects with othercancers). Data represent the fourth subject group (774 subjects). Fordisplay purposes, Ct values are subtracted from 45 (45−Ct). Higher 45−Ctvalues correspond to higher methylation status, demonstratinghypermethylation in subjects with colorectal cancer.

FIG. 27 is a graph representing Ct values from MSRE-qPCR of ADAMTS2 '254for subjects with colorectal cancer and control subjects (healthysubjects, subjects with non-advanced adenoma, and subjects with othercancers). Data represent the fourth subject group (774 subjects). Fordisplay purposes, Ct values are subtracted from 45 (45−Ct). Higher 45−Ctvalues correspond to higher methylation status, demonstratinghypermethylation in subjects with colorectal cancer.

FIG. 28 is a graph representing Ct values from MSRE-qPCR of ADAMTS2 '284for subjects with colorectal cancer and control subjects (healthysubjects, subjects with non-advanced adenoma, and subjects with othercancers). Data represent the fourth subject group (774 subjects). Fordisplay purposes, Ct values are subtracted from 45 (45−Ct). Higher 45−Ctvalues correspond to higher methylation status, demonstratinghypermethylation in subjects with colorectal cancer.

FIG. 29 is a graph representing Ct values from MSRE-qPCR of ZNF542 '502for subjects with colorectal cancer and control subjects (healthysubjects, subjects with non-advanced adenoma, and subjects with othercancers). Data represent the fourth subject group (774 subjects). Fordisplay purposes, Ct values are subtracted from 45 (45−Ct). Higher 45−Ctvalues correspond to higher methylation status, demonstratinghypermethylation in subjects with colorectal cancer.

FIG. 30 is a graph representing Ct values from MSRE-qPCR of LONRF2 '281for subjects with colorectal cancer and control subjects (healthysubjects, subjects with non-advanced adenoma, and subjects with othercancers). Data represent the fourth subject group (774 subjects). Fordisplay purposes, Ct values are subtracted from 45 (45−Ct). Higher 45−Ctvalues correspond to higher methylation status, demonstratinghypermethylation in subjects with colorectal cancer.

FIG. 31 is a graph representing Ct values from MSRE-qPCR of ZNF492 '069for subjects with colorectal cancer and control subjects (healthysubjects, subjects with non-advanced adenoma, and subjects with othercancers). Data represent the fourth subject group (774 subjects). Fordisplay purposes, Ct values are subtracted from 45 (45−Ct). Higher 45−Ctvalues correspond to higher methylation status, demonstratinghypermethylation in subjects with colorectal cancer.

DETAILED DESCRIPTION Screening for Colorectal Cancer

There is a need for improved methods of detecting (e.g., screening for)colorectal cancer, including screening for diagnosis of early-stagecolorectal cancer. Despite recommendations for screening of individuals,e.g., over age 50, colorectal cancer screening programs are oftenineffective or unsatisfactory. Improved colorectal cancer screeningimproves diagnosis and reduces colorectal cancer mortality.

DNA methylation (e.g., hypermethylation or hypomethylation) can activateor inactivate genes, including genes that impact cancer development(see, e.g., FIG. 20). Thus, for example, hypermethylation can inactivateone or more genes that typically act to suppress cancer, causing orcontributing to development of cancer in a sample or subject.

The present disclosure includes the discovery that determination of themethylation status of one or more methylation loci provided herein,and/or the methylation status of one or more DMRs provided herein,and/or the methylation status of one or more methylation sites providedherein, provides screening for colorectal cancer, e.g., with a highdegree of sensitivity and/or specificity. The present disclosureprovides compositions and methods including or relating to colorectalcancer methylation biomarkers that, individually or in various panelscomprising two or more colorectal cancer methylation biomarkers, providefor screening of colorectal cancer, e.g., with a high degree ofspecificity and/or sensitivity.

In various embodiments, a colorectal cancer methylation biomarker of thepresent disclosure is selected from a methylation locus that is orincludes ALK, LONRF2, ADAMTS2, FGF14, DMRT1, ST6GALNAC5, MCIDAS, PDGFD,GSG1L, ZNF492, ZNF568, ZNF542, ZNF471, ZNF132, JAM2, and CNRIP1 (see,e.g., Table 1). In various embodiments, a colorectal cancer DMR isselected from ALK '434, CNRIP1 '232, CNRIP1 '272, LONRF2 '281, LONRF2'387, ADAMTS2 '254, ADAMTS2 '284, ADAMTS2 '328, FGF14 '577, DMRT1 '934,ST6GALNAC5 '456, MCIDAS '855, MCIDAS '003, PDGFD '388, PDGFD '921, GSG1L'861, ZNF492 '499, ZNF492 '069, ZNF568 '252, ZNF568 '405, ZNF542 '525,ZNF542 '502, ZNF471 '527, ZNF471 '558, ZNF471 '662, ZNF132 '268, ZNF132'415, and JAM2 '320 (see, e.g., Table 7)

For the avoidance of any doubt, any methylation biomarker providedherein can be, or be included in, among other things, a colorectalcancer methylation biomarker.

In some embodiments, a colorectal cancer methylation biomarker can be orinclude a single methylation locus. In some embodiments, a colorectalcancer methylation biomarker can be or include two or more methylationloci. In some embodiments, a colorectal cancer methylation biomarker canbe or include a single differentially methylated region (DMR). In someembodiments, a methylation locus can be or include two or more DMRs. Insome embodiments, a methylation biomarker can be or include a singlemethylation site. In other embodiments, a methylation biomarker can beor include two or more methylation sites. In some embodiments, amethylation locus can include two or more DMRs and further include DNAregions adjacent to one or more of the included DMRs.

In some instances, a methylation locus is or includes a gene, such as agene provided in Table 1. In some instances a methylation locus is orincludes a portion of a gene, e.g., a portion of a gene provided inTable 1. In some instances, a methylation locus includes but is notlimited to identified nucleic acid boundaries of a gene.

In some instances, a methylation locus is or includes a coding region ofa gene, such as a coding region of a gene provided in Table 1. In someinstances a methylation locus is or includes a portion of the codingregion of gene, e.g., a portion of the coding region a gene provided inTable 1. In some instances, a methylation locus includes but is notlimited to identified nucleic acid boundaries of a coding region ofgene.

In some instances, a methylation locus is or includes a promoter and/orother regulatory region of a gene, such as a promoter and/or otherregulatory region of a gene provided in Table 1. In some instances amethylation locus is or includes a portion of the promoter and/orregulatory region of gene, e.g., a portion of promoter and/or regulatoryregion a gene provided in Table 1. In some instances, a methylationlocus includes but is not limited to identified nucleic acid boundariesof a promoter and/or other regulatory region of gene. In someembodiments a methylation locus is or includes a high CpG densitypromoter, or a portion thereof.

In some embodiments, a methylation locus is or includes non-codingsequence. In some embodiments, a methylation locus is or includes one ormore exons, and/or one or more introns.

In some embodiments, a methylation locus includes a DNA region extendinga predetermined number of nucleotides upstream of a coding sequence,and/or a DNA region extending a predetermined number of nucleotidesdownstream of a coding sequence. In various instances, a predeterminednumber of nucleotides upstream and/or downstream and be or include,e.g., 500 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40 kb,50 kb, 75 kb, or 100 kb. Those of skill in the art will appreciate thatmethylation biomarkers capable of impacting expression of a codingsequence may typically be within any of these distances of the codingsequence, upstream and/or downstream.

Those of skill in the art will appreciate that a methylation locusidentified as a methylation biomarker need not necessarily be assayed ina single experiment, reaction, or amplicon. A single methylation locusidentified as a colorectal cancer methylation biomarker can be assayed,e.g., in a method including separate amplification (or providingoligonucleotide primers and conditions sufficient for amplification of)of one or more distinct or overlapping DNA regions within a methylationlocus, e.g., one or more distinct or overlapping DMRs. Those of skill inthe art will further appreciate that a methylation locus identified as amethylation biomarker need not be analyzed for methylation status ofeach nucleotide, nor each CpG, present within the methylation locus.Rather, a methylation locus that is a methylation biomarker may beanalyzed, e.g., by analysis of a single DNA region within themethylation locus, e.g., by analysis of a single DMR within themethylation locus.

DMRs of the present disclosure can be a methylation locus or include aportion of a methylation locus. In some instances, a DMR is a DNA regionwith a methylation locus that is, e.g., 1 to 5,000 bp in length. Invarious embodiments, a DMR is a DNA region with a methylation locus thatis equal to or less than 5000 bp, 4,000 bp, 3,000 bp, 2,000 bp, 1,000bp, 950 bp, 900 bp, 850 bp, 800 bp, 750 bp, 700 bp, 650 bp, 600 bp, 550bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100bp, 50 bp, 40 bp, 30 bp, 20 bp, or 10 bp in length. In some embodiments,a DMR is 1, 2, 3, 4, 5, 6, 7, 8 or 9 bp in length.

Methylation biomarkers, including without limitation methylation lociand DMRs provided herein, can include at least one methylation site thatis a colorectal cancer methylation biomarker.

For clarity, those of skill in the art will appreciate that termmethylation biomarker is used broadly, such that a methylation locus canbe a methylation biomarker that includes one or more DMRs, each of whichDRMs is also itself a methylation biomarker, and each of which DMRs caninclude one or more methylation sites, each of which methylation sitesis also itself a methylation biomarker. Moreover, a methylationbiomarker can include two or more methylation loci. Accordingly, statusas a methylation biomarker does not turn on the contiguousness ofnucleic acids included in a biomarker, but rather on the existence of achange in methylation status for included DNA region(s) between a firststate and a second state, such as between colorectal cancer andcontrols.

As provided herein, a methylation locus can be any of one or moremethylation loci each of which methylation loci is, includes, or is aportion of a gene identified in Table 1. In some particular embodiments,a colorectal cancer methylation biomarker includes a single methylationlocus that is, includes, or is a portion of a gene identified inTable 1. For example, in various embodiments, e.g., as described herein,a colorectal cancer methylation biomarker can include a methylationlocus that is, includes, or is a portion of a gene selected from ZNF132,ADAMTS2, ZNF542, LONRF2, ZNF492, FGF14, ST6GALNAC5, PDGFD, ZNF471, JAM2,GSG1L, DMRT1, and MCIDAS.

In some particular embodiments, a colorectal cancer methylationbiomarker includes two or more methylation loci, each of which is,includes, or is a portion of a gene identified in Table 1. In someembodiments, a colorectal cancer methylation biomarker includes 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, methylation loci, each ofwhich is, includes, or is a portion of a gene identified in Table 1.

In some particular embodiments, a colorectal cancer methylationbiomarker includes two or more methylation loci, each of which two ormore methylation loci is, includes, or is a portion of a gene identifiedin any one of Tables 1 to 6. In some particular embodiments, acolorectal cancer methylation biomarker includes two or more methylationloci, each of which two or more methylation loci is, includes, or is aportion of a gene identified in any one of Tables 2 to 6. In someparticular embodiments, a colorectal cancer methylation biomarkerincludes two or more methylation loci, each of which two or moremethylation loci is, includes, or is a portion of a gene identified inTable 1. In some particular embodiments, a colorectal cancer methylationbiomarker includes two methylation loci, each of which two or moremethylation loci is, includes, or is a portion of a gene identified inTable 2. In some particular embodiments, a colorectal cancer methylationbiomarker includes three methylation loci, each of which threemethylation loci is, includes, or is a portion of a gene identified inTable 3. In some particular embodiments, a colorectal cancer methylationbiomarker includes four methylation loci, each of which four methylationloci is, includes, or is a portion of a gene identified in Table 4. Insome particular embodiments, a colorectal cancer methylation biomarkerincludes six methylation loci, each of which six methylation loci is,includes, or is a portion of a gene identified in Table 5. In someparticular embodiments, a colorectal cancer methylation biomarkerincludes eleven methylation loci, each of which eleven methylation lociis, includes, or is a portion of a gene identified in Table 6. Invarious particular embodiments, a colorectal cancer methylationbiomarker or colorectal cancer methylation biomarker panel includes oneor more methylation loci of the present disclosure, but does not includea methylation locus that is, includes, or is a portion of one or more ofFGF14, ZNF471, PDGFD, and ALK.

TABLE 1 Methylation loci identified by gene name Example DNA Region ofHomo sapiens (human) genome SEQ ID NO Gene assembly GRCh38 (hg38) ZNF132ZNF132 chr19, bp 58439728 to SEQ ID NO: 1 58440994 DMRT1 DMRT1 chr9, bp841340 to 968090 SEQ ID NO: 2 ALK ALK chr2, bp 29193215 to 29922286 SEQID NO: 3 JAM2 JAM2 chr21, bp 25637848 to SEQ ID NO: 4 25714704 FGF14FGF14 chr13, bp 101919879 to SEQ ID NO: 5 102403137 MCIDAS MCIDAS chr5,bp 55220951 to SEQ ID NO: 6 55221051 ST6GALNAC5 ST6GALNAC5 chr1, bp76866255 to SEQ ID NO: 7 77063388 LONRF2 LONRF2 chr2, bp 100285667 toSEQ ID NO: 8 100323015 PDGFD PDGFD chr11, bp 104163499 to SEQ ID NO: 9104164026 GSG1L GSG1L chr16, bp 27920615 to SEQ ID NO: 10 28064275ZNF492 ZNF492 chr19, bp 22633051 to SEQ ID NO: 11 22666433 ZNF568 ZNF568chr19, bp 36916312 to SEQ ID NO: 12 36943940 ADAMTS2 ADAMTS2 chr5, bp179118114 to SEQ ID NO: 13 179344392 ZNF542 ZNF542 chr19, bp 56367838 toSEQ ID NO: 14 56370986 ZNF471 ZNF471 chr19, bp 56507245 to SEQ ID NO: 1556508589 CNRIP1 CNRIP1 chr2, bp 68293114 to SEQ ID NO: 16 68320928

TABLE 2 Combination of 2 methylation loci ZNF471 FGF14

TABLE 3 Combination of 3 methylation loci ZNF471 FGF14 PDGFD

TABLE 4 Combination of 4 methylation loci ZNF471 FGF14 PDGFD ADAMTS2

TABLE 5 Combination of 6 methylation loci ZNF471 FGF14 PDGFD ADAMTS2ZNF492 ST6GALNAC5

TABLE 6 Combination of 11 methylation loci ZNF471 FGF14 PDGFD ADAMTS2ZNF492 ST6GALNAC5 ZNF542 LONRF2 ZNF132 CNRIP1 ALK

As provided herein, a colorectal cancer methylation biomarker can be anyof one or more DMRs each of which DMRs is present in a methylation locusthat is, includes, or is a portion of a gene identified in Table 1. Insome particular embodiments, a colorectal cancer methylation biomarkeris or includes a single DMR that is, includes all or a portion of, or ispresent in a gene identified in Table 1. For example, in variousembodiments, a colorectal cancer methylation biomarker can include asingle DMR that is, includes all or a portion of, or is present in agene selected from ALK, CNRIP1, LONRF2, ADAMTS2, FGF14, DMRT1,ST6GALNAC5, MCIDAS, PDGFD, GSG1L, ZNF492, ZNF568, ZNF542, ZNF471,ZNF132, and JAM2.

In some particular embodiments, a colorectal cancer methylationbiomarker includes two or more DMRs, each of which is, includes all or aportion of, or is present in a gene identified in Table 1. In someembodiments, a colorectal cancer methylation biomarker includes 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, DMRs, each of which is,includes all or a portion of, or is present in a gene identified inTable 1.

In some particular embodiments, a colorectal cancer methylationbiomarker includes two or more DMRs, each of which two or more DMRs is,includes all or a portion of, or is present in a gene identified in anyone of Tables 1-6. In some particular embodiments, a colorectal cancermethylation biomarker includes two DMRs, which two DMRs include DMRsthat are, include all or a portion of, or are present in the genesidentified in Table 2. In some particular embodiments, a colorectalcancer methylation biomarker includes three DMRs, which three DMRsinclude DMRs that are, include all or a portion of, or are present inthe genes identified in Table 3. In some particular embodiments, acolorectal cancer methylation biomarker includes four DMRs, which fourDMRs include DMRs that are, include all or a portion of, or are presentin the genes identified in Table 4. In some particular embodiments, acolorectal cancer methylation biomarker includes six DMRs, which sixDMRs include DMRs that are, include all or a portion of, or are presentin the genes identified in Table 5. In some particular embodiments, acolorectal cancer methylation biomarker includes eleven DMRs, whicheleven DMRs include DMRs that are, include all or a portion of, or arepresent in the genes identified in Table 6. In various particularembodiments, a colorectal cancer methylation biomarker or colorectalcancer methylation biomarker panel includes one or more DMRs, but theone or more DMRs do not include a DMR that is, includes all or a portionof, or is present in one or more of FGF14, ZNF471, PDGFD, and ALK.

As provided herein, a colorectal cancer methylation biomarker caninclude any of one or more DMRs, each of which DMRs is, includes all of,or includes a portion of a DMR identified in Table 7, including withoutlimitation DMRs specifically as identified in Table 7. In someparticular embodiments, a colorectal cancer methylation biomarker is orincludes a single DMR that is, includes all of, or includes a portion ofa DMR identified in Table 7, including without limitation a DMRspecifically as identified in Table 7, e.g., a DMR of Table 7 selectedfrom the group of DMRs including, without limitation, ALK '434, CNRIP1'232, CNRIP1 '272, LONRF2 '281, LONRF2 '387, ADAMTS2 '254, ADAMTS2 '284,ADAMTS2 '328, FGF14 '577, DMRT1 '934, ST6GALNAC5 '456, MCIDAS '855,MCIDAS '003, PDGFD '388, PDGFD '921, GSG1L '861, ZNF492 '499, ZNF492'069, ZNF568 '252, ZNF568 '405, ZNF542 '525, ZNF542 '502, ZNF471 '527,ZNF471 '558, ZNF471 '662, ZNF132 '268, ZNF132 '415, and JAM2 '320. Forexample, in various embodiments, a colorectal cancer methylationbiomarker can include a single DMR that is, includes all of, or includesa portion of a DMR selected from LONRF2 '281, LONRF2 '387, ADAMTS2 '254,ADAMTS2 '284, ADAMTS2 '328, FGF14 '577, ST6GALNAC5 '456, PDGFD '388,PDGFD '921, ZNF492 '499, ZNF492 '069, ZNF542 '525, ZNF542 '502, ZNF471'527, ZNF471 '558, and ZNF471 '662.

In some particular embodiments, a colorectal cancer methylationbiomarker includes two or more DMRs, each of which DMRs is, includes allof, or includes a portion of a DMR identified in Table 7, includingwithout limitation DMRs specifically as identified in Table 7. In someembodiments, a colorectal cancer methylation biomarker includes 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, or 28 DMRs, each of which DMRs is, includes all of, orincludes a portion of a DMR identified in Table 7, including withoutlimitation DMRs specifically as identified in Table 7.

In some particular embodiments, a colorectal cancer methylationbiomarker includes two or more DMRs, each of which two or more DMRs is,includes all of, or includes a portion of a DMR identified in any one ofTables 7 to 12, including without limitation DMRs and combinationsthereof specifically as identified in Tables 8 to 12. In some particularembodiments, a colorectal cancer methylation biomarker includes twoDMRs, which two DMRs are the DMRs identified in Table 8. In someparticular embodiments, a colorectal cancer methylation biomarkerincludes three DMRs, which three DMRs are the DMRs identified in Table9. In some particular embodiments, a colorectal cancer methylationbiomarker includes five DMRs, which five DMRs are the DMRs identified inTable 10. In some particular embodiments, a colorectal cancermethylation biomarker includes eight DMRs, which eight DMRs are the DMRsidentified in Table 11. In some particular embodiments, a colorectalcancer methylation biomarker includes fifteen DMRs, which fifteen DMRsare the DMRs identified in Table 12. In various particular embodiments,a colorectal cancer methylation biomarker or colorectal cancermethylation biomarker panel includes one or more DMRs of Table 7, butthe one or more DMRs do not include one or more, or all, DMRs of FGF14,ZNF471, PDGFD, and ALK, e.g., do not include one or more, or all, DMRsof FGF14, ZNF471, PDGFD, and ALK as provided in Table 7.

TABLE 7 Colorectal cancer DMRs gene name chr start site end site widthSEQ ID NO Loci Reference Name ALK 2 29921434 29921541 108 SEQ ID NO: 17ALK ′434 CNRIP1 2 68319232 68319342 111 SEQ ID NO: 18 CNRIP1 ′232 LONRF22 100321281 100321395 115 SEQ ID NO: 19 LONRF2 ′281 LONRF2 2 100322387100322463 77 SEQ ID NO: 20 LONRF2 ′387 ADAMTS2 5 179344254 179344348 95SEQ ID NO: 21 ADAMTS2 ′254 ADAMTS2 5 179344284 179344383 100 SEQ ID NO:22 ADAMTS2 ′284 FGF14 13 102394577 102394651 75 SEQ ID NO: 23 FGF14 ′577DMRT1 9 841934 842046 113 SEQ ID NO: 24 DMRT1 ′934 ST6GALNAC5 1 7686845676868525 70 SEQ ID NO: 25 ST6GALNAC5 ′456 MCIDAS 5 55220855 55220971 117SEQ ID NO: 26 MCIDAS ′855 MCIDAS 5 55221003 55221122 120 SEQ ID NO: 27MCIDAS ′003 PDGFD 11 104163388 104163503 116 SEQ ID NO: 28 PDGFD ′388PDGFD 11 104163921 104164058 138 SEQ ID NO: 29 PDGFD ′921 GSG1L 1628063861 28063964 104 SEQ ID NO: 30 GSG1L ′861 ZNF492 19 2263449922634596 98 SEQ ID NO: 31 ZNF492 ′499 ZNF568 19 36916252 36916371 120SEQ ID NO: 32 ZNF568 ′252 ZNF568 19 36916405 36916476 72 SEQ ID NO: 33ZNF568 ′405 ZNF542 19 56368525 56368610 86 SEQ ID NO: 34 ZNF542 ′525ZNF542 19 56368502 56368591 90 SEQ ID NO: 35 ZNF542 ′502 ZNF471 1956507527 56507675 149 SEQ ID NO: 36 ZNF471 ′527 ZNF471 19 5650755856507675 118 SEQ ID NO: 37 ZNF471 ′558 ZNF471 19 56507662 56507750 89SEQ ID NO: 38 ZNF471 ′662 ZNF132 19 58440268 58440435 168 SEQ ID NO: 39ZNF132 ′268 ZNF132 19 55440415 58440523 109 SEQ ID NO: 40 ZNF132 ′415JAM2 21 25640320 25640399 80 SEQ ID NO: 41 JAM2 ′320 ZNF492 19 2263406922634174 106 SEQ ID NO: 42 ZNF492 ′069 CNRIP1 2 68319272 68319342 71 SEQID NO: 43 CNRIP1 ′272 ADAMTS2 5 179344328 179344412 85 SEQ ID NO: 44ADAMTS2 ′328

TABLE 8 Combination of 2 DMRs gene name chr start site end site ZNF47119  56507558  56507675 FGF14 13 102394577 102394651

TABLE 9 Combination of 3 DMRs gene name chr start site end site ZNF47119 56507558 56507675 FGF14 13 102394577 102394651 PDGFD 11 104163388104163503

TABLE 10 Combination of 5 DMRs gene name chr start site end site ZNF47119 56507558 56507675 FGF14 13 102394577 102394651 PDGFD 11 104163388104163503 ZNF471 19 56507527 56507675 ADAMTS2 5 179344284 179344383

TABLE 11 Combination of 8 DMRs gene name chr start site end site ZNF47119 56507558 56507675 FGF14 13 102394577 102394651 PDGFD 11 104163388104163503 ZNF471 19 56507527 56507675 ADAMTS2 5 179344284 179344383ADAMTS2 5 179344254 179344348 ZNF492 19 22634069 22634174 ST6GALNAC5 176868456 76868525

TABLE 12 Combination of 15 DMRs gene name chr start site end site ZNF47119 56507558 56507675 FGF14 13 102394577 102394651 PDGFD 11 104163388104163503 ZNF471 19 56507527 56507675 ADAMTS2 5 179344284 179344383ADAMTS2 5 179344254 179344348 ZNF492 19 22634069 22634174 ST6GALNAC5 176868456 76868525 ZNF542 19 56368502 56368591 LONRF2 2 100321281100321395 ZNF132 19 58440415 58440523 PDGFD 11 104163921 104164058ZNF132 19 58440268 58440435 CNRIP1 2 68319272 68319342 ALK 2 2992143429921541

In various embodiments, a methylation biomarker can be or include one ormore individual nucleotides (e.g., a single individual cysteine residuein the context of CpG) or a plurality of individual cysteine residues(e.g., of a plurality of CpGs) present within one or more methylationloci (e.g, one or more DMRs) provided herein. Thus, in certainembodiments a methylation biomarker is or includes methylation status ofa plurality of individual methylation sites.

In various embodiments, a methylation biomarker is, includes, or ischaracterized by change in methylation status that is a change in themethylation of one or more methylation sites within one or moremethylation loci (e.g., one or more DMRs). In various embodiments, amethylation biomarker is or includes a change in methylation status thatis a change in the number of methylated sites within one or moremethylation loci (e.g., one or more DMRs). In various embodiments, amethylation biomarker is or includes a change in methylation status thatis a change in the frequency of methylation sites within one or moremethylation loci (e.g., one or more DMRs). In various embodiments, amethylation biomarker is or includes a change in methylation status thatis a change in the pattern of methylation sites within one or moremethylation loci (e.g., one or more DMRs).

In various embodiments, methylation status of one or more methylationloci (e.g., one or more DMRs) is expressed as a fraction or percentageof the one or more methylation loci (e.g., the one or more DMRs) presentin a sample that are methylated, e.g., as a fraction of the number ofindividual DNA strands of DNA in a sample that are methylated at one ormore particular methylation loci (e.g., one or more particular DMRs).Those of skill in the art will appreciate that, in some instances, thefraction or percentage of methylation can be calculated from the ratioof methylated DMRs to unmethylated DMRs for one or more analyzed DMRs,e.g., within a sample.

In various embodiments, methylation status of one or more methylationloci (e.g., one or more DMRs) is compared to a reference methylationstatus value and/or to methylation status of the one or more methylationloci (e.g., one or more DMRs) in a reference sample. In certaininstances, a reference is a non-contemporaneous sample from the samesource, e.g., a prior sample from the same source, e.g., from the samesubject. In certain instances, a reference for the methylation status ofone or more methylation loci (e.g., one or more DMRs) is the methylationstatus of the one or more methylation loci (e.g., one or more DMRs) in asample (e.g., a sample from a subject), or a plurality of samples, knownto represent a particular state (e.g., a cancer state or a non-cancerstate). Thus, a reference can be or include one or more predeterminedthresholds, which thresholds can be quantitative (e.g., a methylationvalue) or qualitative. In certain instances, a reference for methylationstatus of a DMR is the methylation status of a nucleotide or pluralityof nucleotides (e.g., a plurality of contiguous oligonucleotides)present in the same sample that does not include nucleotides of the DMR.Those of skill in the art will appreciate that a reference measurementis typically produced by measurement using a methodology identical to,similar to, or comparable to that by which the non-reference measurementwas taken.

Without wishing to be bound by any particular scientific theory, FIG. 20provides a schematic of one possible mechanism by which hypermethylationor hypomethylation of a regulatory sequence of gene can impactexpression. As shown in FIG. 20, hypomethylation can result in increasedexpression and/or hypermethylation can result in suppression ofexpression. In various instances, increased methylation ofexpress-regulatory regions, such as promoter regions and enhancerregions, as compared to a reference can reduce or silence expression ofan operably linked gene, e.g., of an operably linked gene that typicallyacts to suppress cancer. In various embodiments, decreased methylationof expression-regulatory regions, such as promoter regions and enhancerregions, as compared to a reference can increase expression of anoperably linked gene, e.g., of an operably linked gene having anactivity that contributes to oncogenesis. Without wishing to be bound byany particular scientific theory, DNA methylation may provide a morechemically and biologically stable indicator of cancer status than RNAexpression or protein expression per se.

Methylation is typically thought to be highly tissue-specific, providinga dimension of information not necessarily present in DNA sequenceanalysis.

Methylation events that substantially contribute to oncogenesis canoccur, e.g., in expression-regulatory regions of DNA (e.g., at apromoter region, enhancer region, transcription factor binding site,CTCF-binding site, CpG island, or other sequence) operably linked withcancer-associated genes such as genes that typically act to suppresscancer. Accordingly, inactivation of genes that typically act tosuppress cancer results in or contribute to oncogenesis. Moreover, hypermethylation is typically found at CpG islands.

Cancers

Methods and compositions of the present disclosure are useful forscreening for cancer, particularly colorectal cancer. Colorectal cancersinclude, without limitation, colon cancer, rectal cancer, andcombinations thereof. Colorectal cancers include metastatic colorectalcancers and non-metastatic colorectal cancers. Colorectal cancersinclude cancer located in the proximal part of the colon cancer andcancer located the distal part of the colon.

Colorectal cancers include colorectal cancers at any of the variouspossible stages known in the art, including, e.g., Stage I, Stage II,Stage III, and Stage IV colorectal cancers (e.g., stages 0, I, IIA, IIB,IIC, IIIA, IIIB, IIIC, IVA, IVB, and IVC). Colorectal cancers includeall stages of the Tumor/Node/Metastasis (TNM) staging system. Withrespect to colorectal cancer, T can refer to whether the tumor growninto the wall of the colon or rectum, and if so by how many layers; Ncan refer to whether the tumor has spread to lymph nodes, and if so howmany lymph nodes and where they are located; and M can refer to whetherthe cancer has spread to other parts of the body, and if so which partsand to what extent. Particular stages of T, N, and M are known in theart. T stages can include TX, T0, Tis, T1, T2, T3, T4a, and T4b; Nstages can include NX, N0, N1a, N1b, N1c, N2a, and N2b; M stages caninclude M0, M1a, and M1b. Moreover, grades of colorectal cancer caninclude GX, G1, G2, G3, and G4. Various means of staging cancer, andcolorectal cancer in particular, are well known in the art summarized,e.g., on the world wide web atcancer.net/cancer-types/colorectal-cancer/stages.

In certain instances, the present disclosure includes screening of earlystage colorectal cancer. Early stage colorectal cancers can include,e.g., colorectal cancers localized within a subject, e.g., in that theyhave not yet spread to lymph nodes of the subject, e.g., lymph nodesnear to the cancer (stage N0), and have not spread to distant sites(stage M0). Early stage cancers include colorectal cancers correspondingto, e.g., Stages 0 to II C.

Thus, colorectal cancer s of the present disclosure include, among otherthings, pre-malignant colorectal cancer and malignant colorectal cancer.Methods and compositions of the present disclosure are useful forscreening of colorectal cancer in all of its forms and stages, includingwithout limitation those named herein or otherwise known in the art, aswell as all subsets thereof. Accordingly, the person of skill in artwill appreciate that all references to colorectal cancer provided hereinclude, without limitation, colorectal cancer in all of its forms andstages, including without limitation those named herein or otherwiseknown in the art, as well as all subsets thereof.

Subjects and Samples

A sample analyzed using methods and compositions provided herein can beany biological sample and/or any sample including nucleic acid. Invarious particular embodiments, a sample analyzed using methods andcompositions provided herein can be a sample from a mammal. In variousparticular embodiments, a sample analyzed using methods and compositionsprovided herein can be a sample from a human subject. In variousparticular embodiments, a sample analyzed using methods and compositionsprovided herein can be a sample form a mouse, rat, pig, horse, chicken,or cow.

In various instances, a human subject is a subject diagnosed or seekingdiagnosis as having, diagnosed as or seeking diagnosis as at risk ofhaving, and/or diagnosed as or seeking diagnosis as at immediate risk ofhaving, a cancer such as a colorectal cancer. In various instances, ahuman subject is a subjected identified as a subject in need ofcolorectal cancer screening. In certain instances, a human subject is asubjected identified as in need of colorectal cancer screening by amedical practitioner. In various instances, a human subject isidentified as in need of colorectal cancer screening due to age, e.g.,due to an age equal to or greater than 50 years, e.g., an age equal toor greater than 50, 55, 60, 65, 70, 75, 80, 85, or 90 years. In variousinstances, a human subject is a subject not diagnosed as having, not atrisk of having, not at immediate risk of having, not diagnosed ashaving, and/or not seeking diagnosis for a cancer such as a colorectalcancer, or any combination thereof.

A sample from a subject, e.g., a human or other mammalian subject, canbe a sample of, e.g., blood, blood component, cfDNA, ctDNA, stool, orcolorectal tissue. In some particular embodiments, a sample is anexcretion or bodily fluid of a subject (e.g., stool, blood, lymph, orurine of a subject) or a colorectal cancer tissue sample. A sample froma subject can be a cell or tissue sample, e.g., a cell or tissue samplethat is of a cancer or includes cancer cells, e.g., of a tumor or of ametastatic tissue. In various embodiments, a sample from a subject,e.g., a human or other mammalian subject, can be obtained by biopsy(e.g., fine needle aspiration or tissue biopsy) or surgery.

In various particular embodiments, a sample is a sample of cell-free DNA(cfDNA). cfDNA is typically found in human biofluids (e.g., plasma,serum, or urine) in short, double-stranded fragments. The concentrationof cfDNA is typically low, but can significantly increase underparticular conditions, including without limitation pregnancy,autoimmune disorder, myocardial infraction, and cancer. Circulatingtumor DNA (ctDNA) is the component of circulating DNA specificallyderived from cancer cells. ctDNA can be present in human biofluids boundto leukocytes and erythrocytes or not bound to leukocytes anderythrocytes. Various tests for detection of tumor-derived cfDNA arebased on detection of genetic or epigenetic modifications that arecharacteristic of cancer (e.g., of a relevant cancer). Genetic orepigenetic modifications characteristic of cancer can include, withoutlimitation, oncogenic or cancer-associated mutations in tumor-suppressorgenes, activated oncogenes, hypermethylation, and/or chromosomaldisorders. Detection of genetic or epigenetic modificationscharacteristic of cancer can confirm that detected cfDNA is ctDNA.

cfDNA and ctDNA provide a real-time or nearly real time metric of themethylation status of a source tissue. cfDNA and ctDNA demonstrate ahalf-life in blood of about 2 hours, such that a sample taken at a giventime provides a relatively timely reflection of the status of a sourcetissue.

Various methods of isolating nucleic acids from a sample (e.g., ofisolating cfDNA from blood or plasma) are known in the art. Nucleicacids can be isolated, e.g., without limitation, standard DNApurification techniques, by direct gene capture (e.g., by clarificationof a sample to remove assay-inhibiting agents and capturing a targetnucleic acid, if present, from the clarified sample with a capture agentto produce a capture complex, and isolating the capture complex torecover the target nucleic acid).

Methods of Measuring Methylation Status

Methylation status can be measured by a variety of methods known in theart and/or by methods provided herein. Those of skill in the art willappreciate that a method for measuring methylation status can generallybe applied to samples from any source and of any kind, and will furtherbe aware of processing steps available to modify a sample into a formsuitable for measurement by a given methodology. Methods of measuringmethylation status include, without limitation, methods includingmethylation-status-specific polymerase chain reaction (PCR), methodsincluding nucleic acid sequencing, methods including mass spectrometry,methods including methylation-specific nucleases, methods includingmass-based separation, methods including target-specific capture, andmethods including methylation-specific oligonucleotide primers. Certainparticular assays for methylation utilize a bisulfite reagent (e.g.,hydrogen sulfite ions).

Bisulfite reagents can include, among other things, bisulfite,disulfite, hydrogen sulfite, or combinations thereof, which reagents canbe useful in distinguishing methylated and unmethylated nucleic acids.Bisulfite interacts differently with cytosine and 5-methylcytosine. Intypical bisulfite-based methods, contacting of DNA with bisulfitedeaminates unmethylated cytosine to uracil, while methylated cytosineremains unaffected; methylated cytosines, but not unmethylatedcytosines, are selectively retained. Thus, in a bisulfite processedsample, uracil residues stand in place of, and thus provide anidentifying signal for, unmethylated cytosine residues, while remaining(methylated) cytosine residues thus provide an identifying signal formethylated cytosine residues. Bisulfite processed samples can beanalyzed, e.g., by PCR.

Various methylation assay procedures can be used in conjunction withbisulfite treatment to determine methylation status of a target sequencesuch as a DMR. Such assays can include, among others,Methylation-Specific Restriction Enzyme qPCR, sequencing ofbisulfite-treated nucleic acid, PCR (e.g., with sequence-specificamplification), Methylation Specific Nuclease-assisted Minor-alleleEnrichment PCR, and Methylation-Sensitive High Resolution Melting. Insome embodiments, DMRs are amplified from a bisulfite-treated DNA sampleand a DNA sequencing library is prepared for sequencing according to,e.g., an Illumina protocol or transpose-based Nextera XT protocol. Incertain embodiments, high-throughput and/or next-generation sequencingtechniques are used to achieve base-pair level resolution of DNAsequence, permitting analysis of methylation status.

In various embodiments, methylation status is detected by a methodincluding PCR amplification with methylation-specific oligonucleotideprimers (MSP methods), e.g., as applied to bisulfite-treated sample(see, e.g., Herman 1992 Proc. Natl. Acad. Sci. USA 93: 9821-9826, whichis herein incorporated by reference with respect to methods ofdetermining methylation status). Use of methylation-status-specificoligonucleotide primers for amplification of bisulfite-treated DNAallows differentiation between methylated and unmethylated nucleicacids. Oligonucleotide primer pairs for use in MSP methods include atleast one oligonucleotide primer capable of hybridizing with sequencethat includes a methylation cite, e.g., a CpG. An oligonucleotide primerthat includes a T residue at a position complementary to a cytosineresidue will selectively hybridize to templates in which the cytosinewas unmethylated prior to bisulfite treatment, while an oligonucleotideprimer that includes a G residue at a position complementary to acytosine residue will selectively hybridize to templates in which thecytosine was methylated cytosine prior to bisulfite treatment. MSPresults can be obtained with or without sequencing amplicons, e.g.,using gel electrophoresis. MSP (methylation-specific PCR) allows forhighly sensitive detection (detection level of 0.1% of the alleles, withfull specificity) of locus-specific DNA methylation, using PCRamplification of bisulfite-converted DNA.

Another method that can be used to determine methylation status afterbisulfite treatment of a sample is Methylation-Sensitive High ResolutionMelting (MS-HRM) PCR (see, e.g., Hussmann 2018 Methods Mol Biol.1708:551-571, which is herein incorporated by reference with respect tomethods of determining methylation status). MS-HRM is an in-tube,PCR-based method to detect methylation levels at specific loci ofinterest based on hybridization melting. Bisulfite treatment of the DNAprior to performing MS-HRM ensures a different base composition betweenmethylated and unmethylated DNA, which is used to separate the resultingamplicons by high resolution melting. A unique primer design facilitatesa high sensitivity of the assays enabling detection of down to 0.1-1%methylated alleles in an unmethylated background. Oligonucleotideprimers for MS-HRM assays are designed to be complementary to themethylated allele, and a specific annealing temperature enables theseprimers to anneal both to the methylated and the unmethylated allelesthereby increasing the sensitivity of the assays.

Another method that can be used to determine methylation status afterbisulfite treatment of a sample is Quantitative MultiplexMethylation-Specific PCR (QM-MSP). QM-MSP uses methylation specificprimers for sensitive quantification of DNA methylation (see, e.g.,Fackler 2018 Methods Mol Biol. 1708:473-496, which is hereinincorporated by reference with respect to methods of determiningmethylation status). QM-MSP is a two-step PCR approach, where in thefirst step, one pair of gene-specific primers (forward and reverse)amplifies the methylated and unmethylated copies of the same genesimultaneously and in multiplex, in one PCR reaction. Thismethylation-independent amplification step produces amplicons of up to10⁹ copies per μL after 36 cycles of PCR. In the second step, theamplicons of the first reaction are quantified with a standard curveusing real-time PCR and two independent fluorophores to detectmethylated/unmethylated DNA of each gene in the same well (e.g., 6FAMand VIC). One methylated copy is detectable in 100,000 reference genecopies.

Another method that can be used to determine methylation status afterbisulfite treatment of a sample is Methylation SpecificNuclease-assisted Minor-allele Enrichment (MS-NaME) (see, e.g., Liu 2017Nucleic Acids Res. 45(6):e39, which is herein incorporated by referencewith respect to methods of determining methylation status). Ms-NaME isbased on selective hybridization of probes to target sequences in thepresence of DNA nuclease specific to double-stranded (ds) DNA (DSN),such that hybridization results in regions of double-stranded DNA thatare subsequently digested by the DSN. Thus, oligonucleotide probestargeting unmethylated sequences generate local double stranded regionsresulting to digestion of unmethylated targets; oligonucleotide probescapable of hybridizing to methylated sequences generate localdouble-stranded regions that result in digestion of methylated targets,leaving methylated targets intact. Moreover, oligonucleotide probes candirect DSN activity to multiple targets in bisulfite-treated DNA,simultaneously. Subsequent amplification can enrich non-digestedsequences. Ms-NaME can be used, either independently or in combinationwith other techniques provided herein.

Another method that can be used to determine methylation status afterbisulfite treatment of a sample is Methylation-sensitive SingleNucleotide Primer Extension (Ms-SNuPE™) (see, e.g., Gonzalgo 2007 NatProtoc. 2(8):1931-6, which is herein incorporated by reference withrespect to methods of determining methylation status). In Ms-SNuPE,strand-specific PCR is performed to generate a DNA template forquantitative methylation analysis using Ms-SNuPE. SNuPE is thenperformed with oligonucleotide(s) designed to hybridize immediatelyupstream of the CpG site(s) being interrogated. Reaction products can beelectrophoresed on polyacrylamide gels for visualization andquantitation by phosphor-image analysis. Amplicons can also carry adirectly or indirectly detectable labels such as a fluorescent label,radionuclide, or a detachable molecule fragment or other entity having amass that can be distinguished by mass spectrometry. Detection may becarried out and/or visualized by means of, e.g., matrix assisted laserdesorption/ionization mass spectrometry (MALDI) or using electron spraymass spectrometry (ESI).

Certain methods that can be used to determine methylation status afterbisulfite treatment of a sample utilize a first oligonucleotide primer,a second oligonucleotide primer, and an oligonucleotide probe in anamplification-based method. For instance, the oligonucleotide primersand probe can be used in a method of real-time polymerase chain reaction(PCR) or droplet digital PCR (ddPCR). In various instances, the firstoligonucleotide primer, the second oligonucleotide primer, and/or theoligonucleotide probe selectively hybridize methylated DNA and/orunmethylated DNA, such that amplification or probe signal indicatemethylation status of a sample.

Other bisulfite-based methods for detecting methylation status (e.g.,the presence of level of 5-methylcytosine) are disclosed, e.g., inFrommer (1992 Proc Natl Acad Sci USA. 1; 89(5):1827-31, which is hereinincorporated by reference with respect to methods of determiningmethylation status).

Certain methods that can be used to determine methylation status do notinclude bisulfite treatment of a sample. For instance, changes inmethylation status can be detected by a PCR-based process in which DNAis digested with one or more methylation-sensitive restriction enzymes(MSREs) prior to PCR amplification (e.g., by MSRE-qPCR). Typically,MSREs have recognition sites that include at least one CpG motif, suchthat activity of the MSRE is blocked from cleaving a possiblerecognition site if the site includes 5-methylcytosine. (see, e.g.,Beikircher 2018 Methods Mol Biol. 1708:407-424, which is hereinincorporated by reference with respect to methods of determiningmethylation status). Thus, MSREs selectively digest nucleic acids basedupon methylation status of the recognition site of the MSRE; they candigest DNA at MSRE recognition sites that are unmethylated, but notdigest DNA in MSRE recognition sites that are methylated. In certainembodiments, an aliquot of sample can be digested with MSREs, generatinga processed sample in which unmethylated DNA has been cleaved by theMSREs, such that, the proportion of uncleaved and/or amplifiable DNAwith at least one methylated site within MSRE recognition sites (e.g.,at least one methylated site within each MSRE recognition site of theDNA molecule) is increased relative to uncleaved and/or amplifiable DNAthat did not include at least one methylated site within MSRErecognition sites (e.g., did not include at least one methylated sitewithin each MSRE recognition site of the DNA molecule). Uncleavedsequences of a restriction-enzyme-digested sample can then bepreamplified, e.g, in PCR, and quantified e.g. by qPCR, real-time PCR,or digital PCR. Oligonucleotide primers for MSRE-qPCR amplify regionsthat include one or more MSRE cleavage sites, and/or a plurality of MSREcleavage sites. Amplicons including a plurality of MSRE cleavage sitesare typically more likely to yield robust results. The number ofcleavage sites within a DMR amplicon, and in some instances theresulting robustness of methylation status determination for the DMR,can be increased by design of DMRs that include a plurality of MSRErecognition sites (as opposed to a single recognition site) in a DMRamplicon. In various instances, a plurality of MSREs can be applied tothe same sample, including, e.g., two or more of AciI, Hin6I, HpyCH4IV,and HpaII (e.g., including AciI, Hin6I, and HpyCH4IV). A plurality ofMSREs (e.g., the combination of AciI, Hin6I, HpyCH4IV, and HpaII, or thecombination of AciI, Hin6I, and HpyCH4IV) can provide improved frequencyof MSRE recognition sites within DMR amplicons.

MSRE-qPCR can also include a pre-amplification step following sampledigestion by MSREs but before qPCR in order to improve the amount ofavailable sample, given the low prevalence of cfDNA in blood.

In certain MSRE-qPCR embodiments, the amount of total DNA is measured inan aliquot of sample in native (e.g., undigested) form using, e.g.,real-time PCR or digital PCR.

Various amplification technologies can be used alone or in conjunctionwith other techniques described herein for detection of methylationstatus. Those of skill in the art, having reviewed the presentspecification, will understand how to combine various amplificationtechnologies known in the art and/or described herein together withvarious other technologies for methylation status determination known inthe art and/or provided herein. Amplification technologies include,without limitation, PCR, e.g., quantitative PCR (qPCR), real-time PCR,and/or digital PCR. Those of skill in the art will appreciate thatpolymerase amplification can multiplex amplification of multiple targetsin a single reaction. PCR amplicons are typically 100 to 2000 base pairsin length. In various instances, an amplification technology issufficient to determine methylations status.

Digital PCR (dPCR) based methods involve dividing and distributing asample across wells of a plate with 96-, 384-, or more wells, or inindividual emulsion droplets (ddPCR) e.g., using a microfluidic device,such that some wells include one or more copies of template and othersinclude no copies of template. Thus, the average number of templatemolecules per well is less than one prior to amplification. The numberof wells in which amplification of template occurs provides a measure oftemplate concentration. If the sample has been contacted with MSRE, thenumber of wells in which amplification of template occurs provides ameasure of the concentration of methylated template.

In various embodiments a fluorescence-based real-time PCR assay, such asMethyLight™, can be used to measure methylation status (see, e.g.,Campan 2018 Methods Mol Biol. 1708:497-513, which is herein incorporatedby reference with respect to methods of determining methylation status)MethyLight is a quantitative, fluorescence-based, real-time PCR methodto sensitively detect and quantify DNA methylation of candidate regionsof the genome. MethyLight is uniquely suited for detecting low-frequencymethylated DNA regions against a high background of unmethylated DNA, asit combines methylation-specific priming with methylation-specificfluorescent probing. Additionally, MethyLight can be combined withDigital PCR, for the highly sensitive detection of individual methylatedmolecules, with use in disease detection and screening.

Real-time PCR-based methods for use in determining methylation statustypically include a step of generating a standard curve for unmethylatedDNA based on analysis of external standards. A standard curve can beconstructed from at least two points and can permit comparison of areal-time Ct value for digested DNA and/or a real-time Ct value forundigested DNA to known quantitative standards. In particular instances,sample Ct values can be determined for MSRE-digested and/or undigestedsamples or sample aliquots, and the genomic equivalents of DNA can becalculated from the standard curve. Ct values of MSRE-digested andundigested DNA can be evaluated to identify amplicons digested (e.g.,efficiently digested; e.g., yielding a Ct value of 45). Amplicons notamplified under either digested or undigested conditions can also beidentified. Corrected Ct values for amplicons of interest can then bedirectly compared across conditions to establish relative differences inmethylation status between conditions. Alternatively or additionally,delta-difference between the Ct values of digested and undigested DNAcan be used to establish relative differences in methylation statusbetween conditions.

Methods of measuring methylation status can include, without limitation,massively parallel sequencing (e.g., next-generation sequencing) todetermine methylation state, e.g., sequencing by—synthesis, real-time(e.g., single-molecule) sequencing, bead emulsion sequencing, nanoporesequencing, or other sequencing techniques known in the art. In someembodiments, a method of measuring methylation status can includewhole-genome sequencing, e.g., with base-pair resolution.

In certain particular embodiments, MSRE-qPCR, among other techniques,can be used to determine the methylation status of a colorectal cancermethylation biomarker that is or includes a single methylation locus. Incertain particular embodiments, MSRE-qPCR, among other techniques, canbe used to determine the methylation status of a colorectal cancermethylation biomarker that is or includes two or more methylation loci.In certain particular embodiments, MSRE-qPCR, among other techniques,can be used to determine the methylation status of a colorectal cancermethylation biomarker that is or includes a single differentiallymethylated region (DMR). In certain particular embodiments, MSRE-qPCR,among other techniques, can be used to determine the methylation statusof a colorectal cancer methylation biomarker that is or includes two ormore DMRs. In certain particular embodiments, MSRE-qPCR, among othertechniques, can be used to determine the methylation status of acolorectal cancer methylation biomarker that is or includes a singlemethylation site. In certain particular embodiments, MSRE-qPCR, amongother techniques, can be used to determine the methylation status of acolorectal cancer methylation biomarker that is or includes two or moremethylation sites. In various embodiments, a colorectal cancermethylation biomarker can be any colorectal cancer methylation biomarkerprovided herein. The present disclosure includes, among other things,oligonucleotide primer pairs for amplification of DMRs, e.g., foramplification of DMRs identified in Table 7.

In certain particular embodiments, a cfDNA sample is derived fromsubject plasma and contacted with MSREs that are or include one or moreof AciI, Hin6I, HpyCH4IV, and HpaII (e.g., AciI, Hin6I, and HpyCH4IV).The digested sample can be preamplified with oligonucleotide primerpairs of one or more DMRs, e.g., with one or more oligonucleotide primerpairs provided in Table 13. Digested DNA, e.g., preamplified digestedDNA, can be quantified with qPCR with oligonucleotide primer pairs ofone or more DMRs, e.g., with one or more oligonucleotide primer pairsprovided in Table 13. qPCR ct values can then be determined and used todetermine methylation status of each DMR amplicon.

It will be appreciated by those of skill in the art that oligonucleotideprimer pairs provided in Table 13 can be used in accordance with anycombination of colorectal cancer methylation biomarkers identifiedherein. The skilled artisan will be aware that the oligonucleotideprimer pairs of Table 13 may be individual included or not included in agiven analysis in order to analyze a particular desire combination ofDRMs.

The person of skill in the art will further appreciate that while otheroligonucleotide primer pairs may be used, selection and pairing ofoligonucleotide primers to produce useful DMR amplicons is non-trivialand represents a substantial contribution.

Those of skill in the art will further appreciate that methods,reagents, and protocols for qPCR are well-known in the art. Unliketraditional PCR, qPCR is able to detect the production of amplicons overtime in amplification (e.g., at the end of each amplification cycle),often by use of an amplification-responsive fluorescence system, e.g.,in combination with a thermocycler with fluorescence-detectioncapability. Two common types of fluorescent reporters used in qPCRinclude (i) double-stranded DNA binding dyes that fluorescesubstantially more brightly when bound than when unbound; and (ii)labeled oligonucleotides (e.g., labeled oligonucleotide primers orlabeled oligonucleotide probes).

Those of skill in the art will appreciate that in embodiments in which aplurality of methylation loci (e.g., a plurality of DMRs) are analyzedfor methylation status in a method of screening for colorectal cancerprovided herein, methylation status of each methylation locus can bemeasured or represented in any of a variety of forms, and themethylation statuses of a plurality of methylation loci (preferably eachmeasured and/or represented in a same, similar, or comparable manner) betogether or cumulatively analyzed or represented in any of a variety offorms. In various embodiments, methylation status of each methylationlocus can be measured as a ct value. In various embodiments, methylationstatus of each methylation locus can be represented as the difference inct value between a measured sample and a reference. In variousembodiments, methylation status of each methylation locus can berepresented as a qualitative comparison to a reference, e.g., byidentification of each methylation locus as hypermethylated or nothypermethyated.

In some embodiments in which a single methylation locus is analyzed,hypermethylation of the single methylation locus constitutes a diagnosisthat a subject is suffering from or possibly suffering from colorectalcancer, while absence of hypermethylation of the single methylationlocus constitutes a diagnosis that the subject is likely not sufferingfrom colorectal cancer. In some embodiments, hypermethylation of asingle methylation locus (e.g., a single DMR) of a plurality of analyzedmethylation loci constitutes a diagnosis that a subject is sufferingfrom or possibly suffering from colorectal cancer, while the absence ofhypermethylation at any methylation locus of a plurality of analyzedmethylation loci constitutes a diagnosis that a subject is likely notsuffering from colorectal cancer. In some embodiments, hypermethylationof a determined percentage (e.g., a predetermined percentage) ofmethylation loci (e.g., at least 10% (e.g., at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or 100%)) of a plurality of analyzedmethylation loci constitutes a diagnosis that a subject is sufferingfrom or possibly suffering from colorectal cancer, while the absence ofhypermethylation of a determined percentage (e.g., a predeterminedpercentage) of methylation loci (e.g., at least 10% (e.g., at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or 100%)) of a plurality ofanalyzed methylation loci constitutes a diagnosis that a subject is notlikely suffering from colorectal cancer. In some embodiments,hypermethylation of a determined number (e.g., a predetermined number)of methylation loci (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28DMRs) of a plurality of analyzed methylation loci (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, or 28 DMRs) constitutes a diagnosis that a subject is sufferingfrom or possibly suffering from colorectal cancer, while the absence ofhypermethylation of a determined number (e.g., a predetermined number)of methylation loci (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28DMRs) of a plurality of analyzed methylation loci (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, or 28 DMRs) constitutes a diagnosis that a subject is not likelysuffering from colorectal cancer.

In some embodiments, methylation status of a plurality of methylationloci (e.g., a plurality of DMRs) is measured qualitatively orquantitatively and the measurement for each of the plurality ofmethylation loci are combined to provide a diagnosis. In someembodiments, the qualitative of quantitatively measured methylationstatus of each of a plurality of methylation loci is individuallyweighted, and weighted values are combined to provide a single valuethat can be comparative to a reference in order to provide a diagnosis.To provide but one example of such an approach, support vector machine(SVM) algorithm can be used to analyze the methylation statuses of aplurality of methylation loci of the present disclosure to produce adiagnosis. At least one objective of the support vector machinealgorithm is to identify a hyperplane in an N-dimensional space (N—thenumber of features) that distinctly classifies the data points with theobjective to find a plane that has the maximum margin, i.e. the maximumdistance between data points of both classes. As discussed in thepresent Examples, an SVM model is built on marker values (e.g., ctvalues) derived from a training sample set (e.g., the first subjectgroup and/or the second subject group) that are transformed to supportvector values upon which a prediction is made. In application of the SVMmodel to new samples, samples will be mapped onto vectoral space themodel and categorized as having a probability of belonging to the firstcondition or the second condition, e.g., based on each new sample'slocation relative to the gap between the two conditions. Those of skillin the art will appreciate that, once relevant compositions and methodshave been identified, vector values can be used in conjunction with anSVM algorithm defined by predict( ) function of R-package (see HypertextTransfer Protocol Secure(HTTPS):/cran.r-project.org/web/packages/e1071/index.html, the SVM ofwhich is hereby incorporated by reference) to easily generate aprediction on a new sample. Accordingly, with compositions and methodsfor colorectal cancer diagnosis disclosed herein in hand (and onlythen), generation of a predictive model utilizing algorithm inputinformation in combination to predict( ) function of R-package (seeHypertext Transfer Protocol Secure(HTTPS):/cran.r-project.org/web/packages/e1071/index.html, the SVM ofwhich is hereby incorporated by reference) to provide colorectal cancerdiagnosis would be straightforward. By way of example, one non-limitingexample of SVM vectors for use in diagnosis of colorectal cancer byanalysis of methylation status of a plurality of DMRs provided herein isprovided in Table 17. Those of skill in the art will appreciate that,with the present disclosure in hand, generation of SVM vectors can beaccomplished according to methods provided herein and otherwise known inthe art.

TABLE 13Colorectal cancer DMR oligonucleotide primer pairs, e.g., for MSRE-qPCRLoci gene name Fp_Seq Rp_Seq Fp SEQ ID Rp SEQ ID Reference Name ALKCCTCCTCACCATCATCAGCGCCC GGTACCTCCCGCCGCCTCTGTTC 45 46 ALK ′434 CNRIP1GCGTGCTGGGTTTAATCTTCACCTCAA ACGGCCCGGTCTTTTACAAGGTGG 47 48 CNRIP1 ′232LONRF2 AGGAAGCAAAGTGACCCCTAAGCCT GGTCCGCCTCCCCTACACCT 49 50 LORNF2 ′281LONRF2 CTCTCAGTCCCGCCGGCTTAGGTA GCAAGAGACGCGGACCTGGAGC 51 52 LORNF2 ′387ADAMTS2 CCACTGCGAAGGGAAGGGGCA CCCTGTTAACGCCCCTTCCCGGTT 53 54ADAMTS2 ′254 ADAMTS2 GCGACCCCAGAAAGCCAGCCT AACGGCTGGGGAGTCGCGGA 55 56ADAMTS2 ′284 FGF14 CAACGGAAACTTCCCGCGCTAC CTCGCCGGGGGCTTCGCTAC 57 58FGF14 ′577 DMRT1 CAAAGCGTCTGGGGCGCTAGT ACTTCTTGCTCCCGGCACCCAGGTC 59 60DMRT1 ′934 ST6GALNAC5 CGCTCAGCCGCTCTCCTCTTCTCT AGCGCTAAACACACTGCCAGACCA61 62 ST6GALNAC5 ′456 MCIDAS GGGTTCGGAGCGTGCAAAAGGTGAGAACAGTTCAGTGCATCCCCGCCC 63 64 MCIDAS ′855 MCIDASGCGCCCCACTTACATCCAGCACC ACGTGACATTGACCCAGAAACAGGAGGA 65 66 MCIDAS ′003PDGFD AACGTCTATCACCCAGGGAAAGCT TCCCGGAGTTGGCGAAAGTTGCAA 67 68 PDGFD ′388PDGFD GGTGCATTTGGCATCAGCGACTAGAGAC CATTAGCACAGCGACCCGGGCCAG 69 70PDGFD ′921 GSG1L CCGAAAGAAATCCGAGCCAGGGTGA GGTTTTGTTGCCCCACGTCC 71 72GSG1L ′861 ZNF492 CGAGAGAGGGGAAGGGGCTGGTTG CGAACTTGGGGCGCAGATTGTGG 73 74ZNF492 ′499 ZNF568 GCCCAAGCCTCACCCTCACACAG CGAACCATCCCTCCGCGCCA 75 76ZNF568 ′252 ZNF568 GGTCGCCTTCACCCAGCATCTCAG CAGCGTCACCTGCCGGAAACACC 7778 ZNF568 ′405 ZNF542 CCAGAGGCCCAGGGATCCGTTCAG ACGCGAGCATTCTTGTAAGGCACCC79 80 ZNF542 ′525 ZNF542 GGGAGGAGTGGGCGGCTGAATGG GCACCCGCCACCTCCAAACTCAG81 82 ZNF542 ′502 ZNF471 CCCCACGCGTACTCACACCGAAG GCGGGTAAGAGCAGGAGTGTG83 84 ZNF471 ′527 ZNF471 GTCGCGCGTTTCCCTCCCAG GCGGGTAAGAGCGAGGAGTGTG 8586 ZNF471 ′558 ZNF471 CTGCTCTTACCCGCCGGAACCCTG GAGGGACCTTAGAGCAGAGCGGGC87 88 ZNF471 ′662 ZNF132 CTACTGCTAGGTCGTTGCCAAGGTGATTGGCCAGCGTCTTACACTCCG 89 90 ZNF132 ′268 ZNF132GTGTAAGACGCTGGCCAATCACA ACAACGCGGTCCCTTCAGAAGCAG 91 92 ZNF132 ′415 JAM2CCGCGTGGTCTGGGCTCTGTAG GAATTCCCTCCACCTCCGCCCCAC 93 94 JAM2 ′320 ZNF492CAACGTTAAAGGCAAACACCTTCTGC GGCCGAATGAGGACAGAGTGACAG 95 96 ZNF492 ′069CNRIP1 GCCGGTGAGCAGCTTGATGGT ACGGCCCGGTCTTTTACAAGG 97 98 CNRIP1 ′272ADAMTS2 CGGGAGGGGCGTTAACAGGGC TCTTGGCAGGCAAGGTCTCCGGAG 99 100ADAMTS2 ′328

Applications

Methods and compositions of the present disclosure can be used in any ofa variety of applications. For example, methods and compositions of thepresent disclosure can be used to screen, or aid in screening for,colorectal cancer. In various instances, screening using methods andcompositions of the present disclosure can detect any stage ofcolorectal cancer, including without limitation early-stage colorectalcancer. In some embodiments, colorectal cancer screening using methodsand compositions of the present disclosure is applied to individuals 50years of age or older, e.g., 50, 55, 60, 65, 70, 75, 80, 85, or 90 yearsor older. In some embodiments, colorectal cancer screening using methodsand compositions of the present disclosure is applied to individuals 20years of age or older, e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, or 90 years or older. In some embodiments, colorectal cancerscreening using methods and compositions of the present disclosure isapplied to individuals 20 to 50 years of age, e.g., 20 to 30 years ofage, 20 to 40 years of age, 20 to 50 years of age, 30 to 40 years ofage, 30 to 50 years of age, or 40 to 50 years of age. In variousembodiments, colorectal cancer screening using methods and compositionsof the present disclosure is applied to individuals experiencingabdominal pain or discomfort, e.g., experiencing undiagnosed orincompletely diagnosed abdominal pain or discomfort. In variousembodiments, colorectal cancer screening using methods and compositionsof the present disclosure is applied to individuals experiencing nosymptoms likely to be associated with colorectal cancer. Thus, incertain embodiments, colorectal cancer screening using methods andcompositions of the present disclosure is fully or partiallypreventative or prophylactic, at least with respect to later ornon-early stages of colorectal cancer.

In various embodiments, colorectal cancer screening using methods andcompositions of the present disclosure can be applied to an asymptomatichuman subject. As used herein, a subject can be referred to as“asymptomatic” if the subject does not report, and/or demonstrate bynon-invasively observable indicia (e.g., without one, several, or all ofdevice-based probing, tissue sample analysis, bodily fluid analysis,surgery, or colorectal cancer screening), sufficient characteristics ofcolorectal cancer to support a medically reasonable suspicion that thesubject is likely suffering from colorectal cancer, and/or from cancer.Detection of early stage colorectal cancer is particularly likely inasymptomatic individuals screened in accordance with methods andcompositions of the present disclosure.

In various embodiments, colorectal cancer screening using methods andcompositions of the present disclosure can be applied to a symptomatichuman subject. As used herein, a subject can be referred to as“symptomatic” if the subject report, and/or demonstrates bynon-invasively observable indicia (e.g., without one, several, or all ofdevice-based probing, tissue sample analysis, bodily fluid analysis,surgery, or colorectal cancer screening), sufficient characteristics ofcolorectal cancer to support a medically reasonable suspicion that thesubject is likely suffering from colorectal cancer, and/or from cancer.Symptoms of colorectal cancer can include, without limitation, change inbowel habits (diarrhea, constipation, or narrowing of the stool) thatare persistent (e.g., lasting more than 3 days), feeling of a need tohave a bowel movement which feeling is not relieved upon bowel movement,rectal bleeding (e.g., with bright red blood), blood in stool (which cancause stool to appear dark), abdominal cramping, abdominal pain,weakness, fatigue, unintended weight loss, anemia, and combinationsthereof. Those of skill in the art will appreciate that individualsymptoms that would not alone indicate or raise a suspicion ofcolorectal cancer may do so when presented in combination, e.g., acombination of abdominal cramping and blood in stool, to provide but onenon-limiting example.

Those of skill in the art will appreciate that regular, preventative,and/or prophylactic screening for colorectal cancer improves diagnosisof colorectal cancer, including and/or particularly early stage cancer.As noted above, early stage cancers include, according to at least onesystem of cancer staging, Stages 0 to II C of colorectal cancer. Thus,the present disclosure provides, among other things, methods andcompositions particularly useful for the diagnosis and treatment ofearly stage colorectal cancer. Generally, and particularly inembodiments in which colorectal cancer screening in accordance with thepresent disclosure is carried out annually, and/or in which a subject isasymptomatic at time of screening, methods and compositions of thepresent invention are especially likely to detect early stage colorectalcancer.

In various embodiments colorectal cancer screening in accordance withthe present disclosure is performed once for a given subject or multipletimes for a given subject. In various embodiments, colorectal cancerscreening in accordance with the present disclosure is performed on aregular basis, e.g., every six months, annually, every two years, everythree years, every four years, every five years, or every ten years.

In various embodiments, screening for colorectal cancer using methodsand compositions disclosed herein will be provide a diagnosis ofcolorectal cancer. In other instances, screening for colorectal cancerusing methods and compositions disclosed herein will be indicative ofcolorectal cancer diagnosis but not definitive for colorectal cancerdiagnosis. In various instances in which methods and compositions of thepresent disclosure are used to screen for colorectal cancer, screeningusing methods and compositions of the present disclosure can be followedby a further diagnosis-confirmatory assay, which further assay canconfirm, support, undermine, or reject a diagnosis resulting from priorscreening, e.g., screening in accordance with the present disclosure. Asused herein, a diagnosis-confirmatory assay can be a colorectal cancerassay that provides a diagnosis recognized as definitive by medicalpractitioners, e.g., a colonoscopy-based diagnosed, or a colorectalcancer assay that substantially increases or decreases the likelihoodthat a prior diagnosis was correct, e.g., a diagnosis resulting fromscreening in accordance with the present disclosure.Diagnosis-confirmatory assays could include existing screeningtechnologies, which are generally in need of improvement with respect toone or more of sensitivity, specificity, and non-invasiveness,particularly in the detection of early stage colorectal cancers.

In some instances, a diagnosis-confirmatory assay is a test that is orincludes a visual or structural inspection of subject tissues, e.g., bycolonoscopy. In some embodiments, colonoscopy includes or is followed byhistological analysis. Visual and/or structural assays for colorectalcancer can include inspection of the structure of the colon and/orrectum for any abnormal tissues and/or structures. Visual and/orstructural inspection can be conducted, for example, by use of a scopevia the rectum or by CT-scan. In some instances, adiagnosis-confirmatory assay is a colonoscopy, e.g., including orfollowed by histological analysis. According to some reports,colonoscopy is currently the predominant and/or most relied upondiagnosis-confirmatory assay.

Another visual and/or structural diagnosis confirmatory assay based oncomputer tomography (CT) is CT colonography, sometimes referred to asvirtual colonoscopy. A CT scan utilizes numerous x-ray images of thecolon and/or rectum to produce dimensional representations of the colon.Although useful as a diagnosis-confirmatory assay, some reports suggestthat CT colonography is not sufficient for replacement of colonoscopy,at least in part because a medical practitioner has not physicallyaccessed the subject's colon to obtain tissue for histological analysis.

Another diagnosis-confirmatory assay can be a sigmoidoscopy. Insigmoidoscopy, a sigmoidoscope is used via the rectum to image portionsof the colon and/or rectum. According to some reports, sigmoidoscopy isnot widely used.

In some instances, a diagnosis-confirmatory assay is a stool-basedassay. Typically, stool-based assays, when used in place of visual orstructural inspection, are recommended to be utilized at a greaterfrequency than would be required if using visual or structuralinspection. In some instances, a diagnosis-confirmatory assay is aguiac-based fecal occult blood test or a fecal immunochemical test(gFOBTs/FITs) (see, e.g., Navarro 2017 World J Gastroenterol.23(20):3632-3642, which is herein incorporated by reference with respectto colorectal cancer assays). FOBTs and FITs are sometimes used fordiagnosis of colorectal cancer (see, e.g., Nakamura 2010 J DiabetesInvestig. October 19; 1(5):208-11, which is herein incorporated byreference with respect to colorectal cancer assays). FIT is based ondetection of occult blood in stool, the presence of which is oftenindicative of colorectal cancer but is often not in sufficient volume topermit identification by the unaided eye. For example, in a typical FIT,the test utilizes hemoglobin-specific reagent to test for occult bloodin a stool sample. In various instances, FIT kits are suitable for useby individuals in their own homes. When used in the absence of otherdiagnosis-confirmatory assays, FIT may be recommended for use on anannual basis. FIT is generally not relied upon to provide sufficientdiagnostic information for conclusive diagnosis of colorectal cancer.

Diagnosis-confirmatory assays also include gFOBT, which is designed todetect occult blood in stool by chemical reaction. Like FIT, when usedin the absence of other diagnosis-confirmatory assays, gFOBT may berecommended for use on an annual basis. gFOBT is generally not reliedupon to provide sufficient diagnostic information for conclusivediagnosis of colorectal cancer.

Diagnosis-confirmatory assays can also include stool DNA testing. StoolDNA testing for colorectal cancer can be designed to identify DNAsequences characteristic of cancer in stool samples. When used in theabsence of other diagnosis-confirmatory assays, stool DNA testing may berecommended for use every three years. Stool DNA testing is generallynot relied upon to provide sufficient diagnostic information forconclusive diagnosis of colorectal cancer.

One particular screening technology is a stool-based screening test(Cologuard® (Exact Sciences Corporation, Madison, Wis., United States),which combines an FIT assay with analysis of DNA for abnormalmodifications, such as mutation and methylation. The Cologuard® testdemonstrates improved sensitivity as compared to FIT assay alone, butcan be clinically impracticable or ineffective due to low compliancerates, which low compliance rates are at least in part due to subjectdislike of using stool-based assays (see, e.g., doi:10.1056/NEJMc1405215 (e.g., 2014 N Engl J Med. 371(2):184-188)). TheCologuard® test appears to leave almost half of the eligible populationout of the screening programs (see, e.g., van der Vlugt 2017 Br JCancer. 116(1):44-49). Use of screening as provided herein, e.g., by ablood-based analysis, would increase the number of individuals electingto screen for colorectal cancer (see, e.g., Adler 2014 BMCGastroenterol. 14:183; Liles 2017 Cancer Treatment and ResearchCommunications 10: 27-31). To present knowledge, only one existingscreening technology for colorectal cancer, Epiprocolon, is FDA-approvedand CE-IVD marked and is blood-based. Epiprocolon is based onhypermethylation of SEPT9 gene. The Epiprocolon test suffers from lowaccuracy for colorectal cancer detection with sensitivity of 68% andadvanced adenoma sensitivity of only 22% (see, e.g., Potter 2014 ClinChem. 60(9):1183-91). There is need in the art for, among other things,a non-invasive colorectal cancer screen that will likely achieve highsubject adherence with high and/or improved specificity and/orsensitivity.

In various embodiments, screening in accordance with methods andcompositions of the present disclosure reduces colorectal cancermortality, e.g., by early colorectal cancer diagnosis. Data supportsthat colorectal cancer screening reduces colorectal cancer mortality,which effect persisted for over 30 years (see, e.g., Shaukat 2013 N EnglJ Med. 369(12):1106-14). Moreover, colorectal cancer is particularlydifficult to treat at least in part because colorectal cancer, absenttimely screening, may not be detected until cancer is past early stages.For at least this reason, treatment of colorectal cancer is oftenunsuccessful. To maximize population-wide improvement of colorectalcancer outcomes, utilization of screening in accordance with the presentdisclosure can be paired with, e.g., recruitment of eligible subjects toensure widespread screening.

In various embodiments, screening of colorectal cancer including one ormore methods and/or composition s disclosed herein is followed bytreatment of colorectal cancer, e.g., treatment of early stagecolorectal cancer. In various embodiments, treatment of colorectalcancer, e.g., early stage colorectal cancer, includes administration ofa therapeutic regimen including one or more of surgery, radiationtherapy, and chemotherapy. In various embodiments, treatment ofcolorectal cancer, e.g., early stage colorectal cancer, includesadministration of a therapeutic regimen including one or more oftreatments provided herein for treatment of stage 0 colorectal cancer,stage I colorectal cancer, and/or stage II colorectal cancer.

In various embodiments, treatment of colorectal cancer includestreatment of early stage colorectal cancer, e.g., stage 0 colorectalcancer or stage I colorectal cancer, by one or more of surgical removalof cancerous tissue e.g., by local excision (e.g., by colonoscope),partial colectomy, or complete colectomy.

In various embodiments, treatment of colorectal cancer includestreatment of early stage colorectal cancer, e.g., stage II colorectalcancer, by one or more of surgical removal of cancerous tissue (e.g., bylocal excision (e.g., by colonoscope), partial colectomy, or completecolectomy), surgery to remove lymph nodes near to identified colorectalcancer tissue, and chemotherapy (e.g., administration of one or more of5-FU and leucovorin, oxaliplatin, or capecitabine).

In various embodiments, treatment of colorectal cancer includestreatment of stage III colorectal cancer, by one or more of surgicalremoval of cancerous tissue (e.g., by local excision (e.g., bycolonoscopy-based excision), partial colectomy, or complete colectomy),surgical removal of lymph nodes near to identified colorectal cancertissue, chemotherapy (e.g., administration of one or more of 5-FU,leucovorin, oxaliplatin, capecitabine, e.g., in a combination of (i)5-FU and leucovorin, (ii) 5-FU, leucovorin, and oxaliplatin (e.g.,FOLFOX), or (iii) capecitabine and oxaliplatin (e.g., CAPEOX)), andradiation therapy.

In various embodiments, treatment of colorectal cancer includestreatment of stage IV colorectal cancer, by one or more of surgicalremoval of cancerous tissue (e.g., by local excision (e.g., bycolonoscope), partial colectomy, or complete colectomy), surgicalremoval of lymph nodes near to identified colorectal cancer tissue,surgical removal of metastases, chemotherapy (e.g., administration ofone or more of 5-FU, leucovorin, oxaliplatin, capecitabine, irinotecan,VEGF-targeted therapeutic agent (e.g., bevacizumab, ziv-aflibercept, orramucirumab), EGFR-targeted therapeutic agent (e.g., cetuximab orpanitumumab), Regorafenib, trifluridine, and tipiracil, e.g., in acombination of or including (i) 5-FU and leucovorin, (ii) 5-FU,leucovorin, and oxaliplatin (e.g., FOLFOX), (iii) capecitabine andoxaliplatin (e.g., CAPEOX), (iv) leucovorin, 5-FU, oxaliplatin, andirinotecan (FOLFOXIRI), and (v) trifluridine and tipiracil (Lonsurf)),radiation therapy, hepatic artery infusion (e.g., if cancer hasmetastasized to liver), ablation of tumors, embolization of tumors,colon stent, colorectomy, colostomy (e.g., diverting colostomy), andimmunotherapy (e.g., pembrolizumab).

Those of skill in the art that treatments of colorectal cancer providedherein can be utilized, e.g., as determined by a medical practitioner,alone or in any combination, in any order, regimen, and/or therapeuticprogram. Those of skill in the art will further appreciate that advancedtreatment options may be appropriate for earlier stage cancers insubjects previously having suffered a cancer or colorectal cancer, e.g.,subjects diagnosed as having a recurrent colorectal cancer.

In some embodiments, methods and compositions for colorectal cancerscreening provided herein can inform treatment and/or payment (e.g.,reimbursement for or reduction of cost of medical care, such asscreening or treatment) decisions and/or actions, e.g., by individuals,healthcare facilities, healthcare practitioners, health insuranceproviders, governmental bodies, or other parties interested inhealthcare cost.

In some embodiments, methods and compositions for colorectal cancerscreening provided herein can inform decision making relating to whetherhealth insurance providers reimburse a healthcare cost payer orrecipient (or not), e.g., for (1) screening itself (e.g., reimbursementfor screening otherwise unavailable, available only for periodic/regularscreening, or available only for temporally- and/orincidentally-motivated screening); and/or for (2) treatment, includinginitiating, maintaining, and/or altering therapy, e.g., based onscreening results. For example, in some embodiments, methods andcompositions for colorectal cancer screening provided herein are used asthe basis for, to contribute to, or support a determination as towhether a reimbursement or cost reduction will be provided to ahealthcare cost payer or recipient. In some instances, a party seekingreimbursement or cost reduction can provide results of a screenconducted in accordance with the present specification together with arequest for such reimbursement or cost reduction of a healthcare cost.In some instances, a party making a determination as to whether or notto provide a reimbursement or cost reduction of a healthcare cost willreach a determination based in whole or in part upon receipt and/orreview of results of a screen conducted in accordance with the presentspecification.

For the avoidance of any doubt, those of skill in the art willappreciate from the present disclosure that methods and compositions forcolorectal cancer diagnosis of the present specification are at leastfor in vitro use. Accordingly, all aspects and embodiments of thepresent disclosure can be performed and/or used at least in vitro.

Kits

The present disclosure includes, among other things, kits including oneor more compositions for use in colorectal cancer screening as providedherein, optionally in combination with instructions for use thereof incolorectal cancer screening. In various embodiments, a kit for screeningof colorectal cancer can include one or more of: one or moreoligonucleotide primers (e.g., one or more oligonucleotide primer pairs,e.g., as found in Table 13), one or more MSREs, one or more reagents forqPCR (e.g., reagents sufficient for a complete qPCR reaction mixture,including without limitation dNTP and polymerase), and instructions foruse of one or more components of the kit for colorectal cancerscreening. In various embodiments, a kit for screening of colorectalcancer can include one or more of: one or more oligonucleotide primers(e.g., one or more oligonucleotide primer pairs, e.g., as found in Table13), one or more bisulfite reagents, one or more reagents for qPCR(e.g., reagents sufficient for a complete qPCR reaction mixture,including without limitation dNTP and polymerase), and instructions foruse of one or more components of the kit for colorectal cancerscreening.

In certain embodiments, a kit of the present disclosure includes atleast one oligonucleotide primer pair for amplification of a methylationlocus and/or DMR as disclosed herein.

In some instances, a kit of the present disclosure includes one or moreoligonucleotide primer pairs for amplification of one or moremethylation loci of the present disclosure. In some instances, kit ofthe present disclosure includes one or more oligonucleotide primer pairsfor amplification of one or more methylation loci that are or includeall or a portion of one or more genes provided in Table 1. In someparticular instances, a kit of the present disclosure includesoligonucleotide primer pairs for a plurality of methylation loci thateach are or include all or a portion of a gene identified in Table 1,the plurality of methylation loci including, e.g., 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 methylation loci, e.g., as provided in anyof Tables 1 to 6.

In some instances, a kit of the present disclosure includes one or moreoligonucleotide primer pairs for amplification of one or more DMRs ofthe present disclosure. In some instances, kit of the present disclosureincludes one or more oligonucleotide primer pairs for amplification ofone or more DMRs that are, include all or a portion of, or are within agene identified in Table 1. In some particular embodiments, a kit of thepresent disclosure includes oligonucleotide primer pairs for a pluralityof DMRs each of which is, includes all or a portion of, or is within agene identified in Table 1, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16 DMRs, e.g., in accordance with any one of Tables 1 to6.

In some instances, kit of the present disclosure includes one or moreoligonucleotide primer pairs for amplification of one or more DMRs ofTable 7. In some particular instances, a kit of the present disclosureincludes oligonucleotide primer pairs for a plurality of DMRs of Table7, the plurality of DMRs including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or28 DMRs of Table 7, e.g., as provided in any of Tables 8 to 12.

In various embodiments, a kit of the present disclosure includes one ormore oligonucleotide primer pairs provided in Table 13. Those of skillin the art will appreciate that oligonucleotide primer pairs provided inTable 13 can be provided in any combination of one or moreoligonucleotide primer pairs, e.g., in a combination as provided in anyone of Tables 1-12.

In various particular embodiments, kit of the present disclosure doesnot include oligonucleotide primer pairs that amplify all or a portionof one or more of FGF14, ZNF471, PDGFD, and ALK.

A kit of the present disclosure can further include one or more MSREsindividually or in a single solution. In various embodiments, one ormore MSREs are selected from the set of MSREs including AciI, Hin6I,HpyCH4IV, and HpaII (e.g., such that the kit includes AciI, Hin6I, andHpyCH4IV, either individually or in a single solution). In certainembodiments, a kit of the present disclosure includes one or morereagents for qPCR (e.g., reagents sufficient for a complete qPCRreaction mixture, including without limitation dNTP and polymerase).

EXAMPLES

The present Examples confirm that the present disclosure providesmethods and compositions for, among other things, screening for andtreatment of colorectal cancer. The present Examples further demonstratethat compositions and methods provided herein provide a remarkably highdegree of sensitivity and specificity in screening and/or treatment ofcolorectal cancer. Also provided are clinical studies comparingmethylation of biomarkers in samples from subjects diagnosed as havingcolorectal cancer and methylation of biomarkers in samples from controlsubjects, further demonstrating screening for colorectal cancerincluding methods and/or compositions of the present disclosure. Exceptas specifically stated otherwise, samples of the present Examples arehumans or of human origin. With the exception of Example 1, allexperiments were performed using plasma samples.

Example 1. Identification of Methylation Biomarkers Associated withColorectal Cancer

The present Example includes identification of CpG loci that arehypermethylated in one or more of colon cancer and rectal cancer ascompared to healthy controls. In particular, experiments of the presentExample examined CpG methylation in samples from (i) colon cancers of341 subjects previously diagnosed as suffering from colon cancer, whichsubjects had not been previously treated by chemotherapy orradiotherapy; (ii) rectal cancers of 118 subjects previously diagnosedas suffering from rectal cancer, which subjects had not been previouslytreated by chemotherapy or radiotherapy; (iii) colons of 40 healthycontrol subjects not diagnosed as suffering from colorectal cancer; and(iv) leukocytes of 10 healthy control subjects not diagnosed assuffering from a colorectal cancer. Tissue samples were of fresh frozentissue.

Samples were analyzed for DNA methylation by a global methylomicsanalysis platform (Infinium HumanMethylation450 (HM450) beadarray). TheInfinium HumanMethylation450 array assesses methylation status of >450000 CpGs located throughout the genome. DNA methylation profiles wereobtained from all the tissue samples.

CpG methylation sites for which methylation status did not substantiallydiffer between a colorectal cancer and healthy controls were identifiedand removed from consideration (mean b-value <0.25 and b-value >0.3 inno more than five samples across the entire set). This filteringproduced a list of CpG methylation sites for which methylation statussubstantially differed between a colorectal cancer and healthy controls.The resulting set of CpG methylation sites was then further filtered byexcluding CpG methylation sites with a mean b-value difference equal toor less than 0.1, yielding 253 CpG methylation sites. Each of the 253CpG methylation sites was associated with a hypermethylated status incolorectal cancer status as compared to controls.

Thus, the present Example generated a set of 253 individual CpGmethylation sites that are methylation biomarkers for colorectal cancer.The 253 methylation biomarkers represent a plurality of DMRs togetherfound within 36 genes, i.e., within 36 methylation loci. Each of the 36DMRs is hypermethylated in colorectal cancer as compared to healthycontrols.

Example 2: Development of Cell-Free DNA Assay for Methylation Biomarkersby MSRE-qPCR

The present Example develops an assay for determining the methylationstatus of colorectal cancer methylation biomarkers based on circulatingcell free DNA (cfDNA. cfDNA is incomplete and fragmented, and themechanism by which the cfDNA is transmitted from cancer cells to blood(as a portion called circulating tumor DNA) is unknown. At least becausethe 253 methylation biomarkers of Example 1 were identified from tissuesamples, it was not known prior to the experiments of the presentExample whether identified colorectal cancer methylation biomarkerscould be sufficiently analyzed from cfDNA to successfully capture thectDNA portion that allows for identifying subjects or samples forcolorectal cancer.

As a critical step toward determining whether colorectal cancermethylation biomarkers identified in Example 1 could be sufficientlyanalyzed from cfDNA to successfully capture the ctDNA portion thatallows for identification of subjects or samples for colorectal cancer,a sensitive assay was developed for screening of these biomarkers. Inparticular, a Methylation-Sensitive Restriction Enzyme (MSRE)-qPCRmethodology was developed. The MSRE-qPCR methodology was developed tomeasure methylation of DMRs covering identified CpG sites in bloodsamples, in particular in cell-free DNA (cfDNA) of tumors present inblood.

Development of the MSRE-qPCR methodology was significant at least inpart because analyzing CpG methylation biomarkers derived from tumortissue by analysis of cfDNA is challenging due to the low concentrationof tumor-derived DNA circulating in blood (0.1-1%) as compared to thenon-tumor DNA background of the sample. Thus, while it is generallypreferred to develop biomarker analyses that rely on readily obtainablesamples such as blood, urine, or stool, use of blood for analysis oftumor derived methylation biomarkers is challenging. Thus, even afteridentification of methylation biomarkers characteristic of colorectalcancer in tissue, as discussed above, it cannot be predicted whether thefragmented and poorly understood nature of ctDNA will permit successfulscreening using methylation biomarkers identified in tissue.

MSRE-qPCR requires design of oligonucleotide primers (MSRE-qPCRoligonucleotide primer pairs) that amplify loci that each include atleast one colorectal cancer MSRE cleavage site (i.e., an MSRE cleavagesite that covers at least one colorectal cancer methylation biomarkersite, such that cleavage of the MSRE cleavage site is permitted innucleic acid molecules where all of the at least one colorectal cancermethylation biomarker sites are unmethylated and blocked in nucleic acidmolecules where at least one of the at least one colorectal cancermethylation biomarker sites is methylated). MSRE-qPCR assays can utilizemultiple restriction enzymes to enhance the range of colorectal cancermethylation biomarker sites that can be assayed by a single MSRE-qPCRreaction, as a single MSRE is unlikely to cleavage sites that togetherinclude all methylation biomarker sites of interest. MSRE-qPCR assays ofthe present Examples utilize the MSREs AciI, Hin6I, and HpyCH4IV, whichtogether were found to provide sufficient coverage.

An exemplary schematic work flow for MSRE-qPCR is provided in FIG. 1. Asperformed in the present Examples, circulating cell-free tumor DNA wasextracted from subject blood (typically a plasma sample of approximately4 mL) by QIAamp MinElute ccfDNA Kit in accordance with manufacturerprotocol (QIAamp MinElute ccfDNA Handbook August 2018, Qiagene). Asshown in FIG. 1, isolated cfDNA was divided into two aliquots, a firstof which aliquots is utilized in a qPCR quality control analysis, and asecond of which aliquots is used in MSRE-qPCR

For MSRE-qPCR, ⅔ of eluted cfDNA by volume was digested with MSREs.Because non-methylated DNA is selectively cleaved, contacting the cfDNAwith the MSREs enriches the sample for methylation-derived signal;methylated DNA remains intact and quantifiable. The remaining ⅓ ofeluted cfDNA by volume was used for qPCR using the MSRE-qPCRoligonucleotide primers to confirm that amplicons were successfullyamplified from cfDNA, which amplification confirms that template ispresent, hence providing technical quality control.

As applied herein, MSRE-qPCR oligonucleotide primer pairs weresuccessfully developed for amplification of DMRs together including 180of the 253 CpG methylation biomarker sites identified in Example 1. DMRstypically included 1 to 15 MSRE cleavage sites, which MSRE cleavagesites together covered each of the 180 methylation biomarker sites. Asapplied herein, methylation status of six genes (JUB, H19, TBP, TCEB2,SNRPN, IRF4) provided a methylation control, which control permittedmonitoring of assay robustness and reproducibility.

Example 3: MSRE-qPCR of cfDNA Successfully Distinguishes Subjects byColorectal Cancer Status

To probe clinical diagnostic and prognostic power of identifiedmethylation biomarkers, the DMRs amplified by the MSRE-qPCRoligonucleotide primer pairs covering the 180 methylation biomarkersites, and appropriate controls, were assayed in cfDNA extracted fromplasma of human subjects. Subjects were undiagnosed individuals seeking,or in the process of obtaining, a diagnosis regarding possiblecolorectal cancer, such that methylation biomarker analysis could beperformed prior to traditional diagnostic testing for colorectal cancerand then compared to a subsequent traditional diagnosis. In particular,cfDNA was sampled from undiagnosed individuals seeking, or in theprocess of obtaining, a diagnosis regarding possible colorectal cancerat screening centers and oncology clinics in Spain and the United Statesbetween 2017 and 2018. A first subject group included 70 suchindividuals (see description of the first subject group in FIG. 2), anda second subject group included 63 such individuals (see description ofsecond subject group in FIG. 3). Initial results based on MSRE-qPCRanalysis of a small panel of tested DMRs of genes shown in Table 14 inthe second subject group provided proof-of-principle for colorectalcancer diagnosis: results demonstrated overall diagnostic sensitivity of80%, for colorectal cancer, with diagnostic sensitivity of up to 75% forearly localized colorectal cancer, and 90% specificity (FIG. 4). Therepresentative proof-of-principle panel of DMRs performed similarly,and/or statistically equally, well with respect to proximal cancers anddistal cancers (FIG. 4). The proof-of-principle panel of DMRs alsoperformed similarly, and/or statistically equally, well with respect tolocalized cancer and advanced cancer (FIG. 4). Moreover, MSRE-qPCRanalysis of methylation of MSRE-qPCR control genes and of undigested DNAcontrols showed high technical reliability of the developed MSRE-qPCRassays in measuring colorectal cancer biomarker methylation status inplasma cfDNA.

FIGS. 5-9 show the association of methylation status of colorectalcancer DMRs with colorectal cancer. Results are displayed as theMSRE-qPCR Ct value subtracted from 45 (i.e., 45−Ct value) for displaypurposes. Results demonstrate the surprisingly high predictive power ofindividual colorectal cancer methylation biomarkers, or of as few asthree individual colorectal cancer methylation biomarkers, of thepresent disclosure for colorectal cancer, e.g., for use in determiningscreening for colorectal cancer in a subject.

TABLE 14 Proof-of-principle DMR panel gene chr start end width uid PDGFDchr11 104163499 104164026 528 PDGFD_chr11_104163499_104164026 FGF14chr13 101919879 102403137 483259 FGF14_chr13_101919879_102403137 ALKchr19 58439728 58440994 1267 ALK_chr19_58439728_58440994 LONRF2 chr229193215 29922286 729072 LONRF2_chr2_29193215_29922286 JAM2 chr268293114 68320928 27815 JAM2_chr2_68293114_68320928

Example 4. Further Validation of Methylation Biomarkers by MSRE-qPCR

To verify the predictive power of methylation biomarker DMRs forcolorectal cancer, data derived from MSRE-qPCR analysis of samples fromthe 133 subjects of the first and second subject groups identified inExample 3 (see FIGS. 2 and 3) were further analyzed. Monte-Carlocross-validation was used over 50 runs and random forest algorithm wasused for feature ranking and markers with VIP>2 were used for building asupport-vector machine (SVM) algorithm-based classification model. Thisanalysis identified several subsets of markers (2, 3, 5, 8, 15, 28 asdescribed in Tables 7-12) that in SVM-model gave a good prediction.

All models (2, 3, 5, 8, 15 and 28 colorectal cancer DMR panels) wereapplied to cfDNA extracted from plasma of a third subject group. Thethird subject group included 82 subjects who had either previouslyreceived a confirmed diagnosis of colorectal cancer or were controlsubjects known to not have colorectal cancer deemed (the control groupincluding subjects having hyperplastic polyps and/or non-advancedadenoma, but not colorectal cancer), based on colonoscopy screening. The82 subjects were subjects attending colorectal cancer screening andoncology units in Spain and the United States. Further description ofthe third subject group is shown in FIG. 10.

Oligonucleotide primer pairs (Table 13) for amplification of the 28 DRMsin MSRE-qPCR cover at least one MSRE cleavage site, typically 3 to 15MSRE cleavage sites. MSRE-qPCR was carried out according to themethodology described in Example 2.

Notwithstanding the sufficiency and utility of all tested panels forscreening for colorectal cancer, those of skill in the art willappreciate that the 28-DMR panel provided increased sensitivity andcomparable specificity as compared to all other DMR panels indicated inTable 15. For the avoidance of any doubt, all of the panels tested,e.g., as described in Tables 7-14, are individually alone sufficient(e.g., in both sensitivity and specificity), and useful, for clinicalscreening of colorectal cancer. Analysis of the third subject groupusing the 28 colorectal cancer DMR panel showed general sensitivity fordiagnosis of colorectal cancer of 79%, with 75% sensitivity forlocalized (early) cancer and 84% sensitivity for advanced cancer. Dataalso revealed specificity of 87% at AUC 82% (FIG. 11). A ROC curveanalysis of the second validation group data based on a 28-marker panelidentified by the SVM model is provided in FIG. 11.

Thus, evaluation of the performance of the 28 colorectal cancer DMRpanel and subsets thereof reveal that both the full panel and each ofthe various subsets of 2, 3, 5, 8, and 15 of the 28 colorectal cancerDMRs are individually sufficient for clinical screening of colorectalcancer (See Tables 7-14). For instance, to highlight just one example,the 3-DMR subset (Table 9) achieved good separation of colorectal cancersubjects from control subjects, demonstrating sufficient performance forclinical screening of colorectal cancer, at least in part asdemonstrated by the determined sensitivity of 60% and specificity of 87%(Table 15).

SVM-model characteristics are described in Table 16. Input support SVMVectors and their coefficiency (weight) values are given in Table 17(due to the size of Table 17, Table 17 is presented in several portions,with the coefficients and gene names repeated in each portion forreference). For prediction purposes the provided information was used incombination with predict( ) function in R-package (see HypertextTransfer Protocol Secure(HTTPS)://cran.r-project.org/web/packages/e1071/index.html).

TABLE 15 Accuracy metrics for application of 28 colorectal cancer DMRpanel and subsets thereof to third subject group 2 3 5 8 15 28 AUC 0.770.82 0.83 0.83 0.82 0.82 AUC_CI_LOW 0.67 0.73 0.73 0.74 0.73 0.72AUC_CI_HIGH 0.87 0.91 0.92 0.92 0.92 0.92 Sensitivity 0.63 0.60 0.650.53 0.72 0.79 Specificity 0.79 0.87 0.82 0.87 0.79 0.87

TABLE 16 SVM-model input characteristics vars value type 0 kernel 2 cost1 degree 3 gamma 0.035714 coef0 0 nu 0.5 epsilon 0.1 nclasses 2 rho−0.73551 probA −2.14936 probB −0.10114 sigma 0

TABLE 17 SVM Vectors _ GSG1L 7NF492_2 ZNF568_2 ZNF568_1 ZNF542_2 coefsGSG1L ′861 7NF492 ′499 ZNF568 ′252 ZNF568 ′405 ZNF542 ′525   1  0.166333806 0.423405118 −0.939531248 0.275236404 −0.052862411   1  0.183718034 −2.19255911 −0.092363545 −0.327541264 0.309450278  0.160992568 −0.094429617 −2.19255911 −0.939531248 −0.4136523590.137466406   1   0.475386751 0.318930431 −0.147777884 0.0199785140.164983826   1 −0.152377044 0.473593938 1.146062978 0.4290062170.574305439   1   0.315065536 0.442866089 0.835406834 0.0415062880.55940017   0.539110203 −0.428593114 0.344536972 0.864793226 −0.05075560.174156299   1   0.143154835 −2.19255911 0.848840613 −0.3736722080.025103611   0.228841426 −0.16396653 0.392677269 −0.939531248−0.0507556 0.161544148   1   0.691723813 0.288202582 −0.9395312480.232180856 0.641952428   1   0.152812739 0.548365038 0.8975380630.321367348 0.6259006   0.086502196   0.152812739 0.413162502−0.939531248 −0.318315075 0.260148235   1   0.058165275 0.455157229−0.939531248 −0.035378619 0.421813073   0.022981588 −0.055797999−2.19255911 −0.939531248 −0.469009492 0.310596837   0.199753156  0.029191561 0.425453641 −0.939531248 −0.192223828 0.392002536  0.640672506   0.401986676 0.512515881 0.731295044 −0.352144434−2.050168434   0.488436741   0.11224954 −2.19255911 −0.939531248−0.327541264 −2.050168434   0.290813367   0.038849466 0.4551572290.842963334 0.038430892 0.370217912   0.93438076 −0.1658981110.292299628 0.996612184 −0.293711905 0.182182213   1   0.345970830.531976852 −0.939531248 0.622756182 0.534175869   0.177133374  0.291886565 0.201140342 0.931122511 0.312141159 0.439011461  0.363673626 −0.252819252 0.199091819 −0.939531248 −0.2322039790.408054364   0.372869951 −0.022961123 −2.19255911 −0.939531248−0.438255529 −2.050168434   0.180416799 −0.0152348 0.246207854−0.939531248 −0.247580961 0.130587052   1   1.837151292 1.131169911−0.939531248 1.084065621 1.180835225   0.236974727 −4.542860450.032137172 −0.240135117 −0.619703909 −2.050168434   0.433173404−4.54286045 −2.19255911 −0.939531248 −0.40442617 0.427545869  0.037394649   0.15474432 −2.19255911 −0.939531248 −0.2045254130.471115116   1   0.245528623 0.455157229 −0.939531248 −0.2076008090.380536944   0.263636311 −4.54286045 −2.19255911 −0.939531248−1.72992196 −2.050168434   1   0.682065908 0.557583393 1.0343946890.828807731 0.838014042   1 −0.042276933 0.55553487 0.7757944380.158371346 0.349579848   1   0.025328399 0.467448369 0.848001002−0.524366624 0.018224256   0.26169536 −4.54286045 −2.19255911−0.939531248 −7.963750185 −2.050168434   0.1799778   0.2957497270.267717349 −0.939531248 0.226030064 0.467675439   0.898868481−0.18521392 0.324051739 −0.939531248 −0.023077034 0.29339845  0.042908076   0.019533657 0.191921987 −0.939531248 −0.6227793050.275053503   0.140898434 −4.54286045 0.204213127 −0.939531248−0.567422172 0.123707697   0.140415448   0.293818146 −2.19255911−0.939531248 −0.699664211 0.061793503   1   0.166333806 0.135587597−0.939531248 −0.044604807 0.459649525   0.108835219 −0.3783720110.333270094 −0.939531248 −0.604326927 −2.050168434   1   0.6086658340.370143513 0.901736119 0.641208559 0.750875547 −1   0.322791859−2.19255911 1.066299914 0.186049912 0.550227697 −1   0.025328399−2.19255911 −0.497895756 0.026129307 0.433278665 −1   0.3112023740.387555961 −0.939531248 −0.182997639 0.5192706 −1 −0.0596611610.322003216 1.020121298 0.422855425 0.403468127 −1   0.4039182570.612893521 0.959669292 0.468986368 0.589210708 −1   0.1721285480.145830214 0.701908652 0.182974516 0.491753181 −1 −0.163966530.411113978 −0.939531248 −0.336767452 0.473408235 −1   0.3363129260.694834453 −0.939531248 0.819581542 0.740556515 −0.27309104  1.360050807 0.923244798 1.68509337 2.378807448 1.22669759 −1  2.101777876 1.346264856 1.939495565 3.255295383 1.550027268 −1  0.712971203 0.404968409 0.776634049 0.475137161 −2.050168434−0.675668601   0.382670867 0.677422005 1.048668079 0.5797006340.674056084 −1   0.245528623 0.403944147 −0.939531248 −0.545894398−2.050168434 −0.228886868   1.516508861 0.993918852 1.7136401512.252716201 1.264534042 −1   0.523676274 0.505346049 0.9227263990.272161008 0.602969418 −0.253709663   0.53719734 0.671276435−0.939531248 0.186049912 0.764634257 −1 −0.007508476 −2.19255911−0.939531248 −0.284485716 0.336967697 −1   0.465728846 0.297420936−0.939531248 0.075335647 0.648831783 −1   0.206897005 0.524807020.981499183 0.066109458 0.583477912 −1   0.626050062 0.583189934−0.939531248 0.804204561 0.654564579 −1   0.094865312 0.3568281110.968065404 0.112240402 0.397735331 −1   0.04850737 −2.19255911−0.939531248 −1.041033197 0.29339845 −1 −0.241229766 −2.192559110.913490675 −0.14916828 0.136319847 −1   0.419370905 −2.19255911−0.939531248 0.099938817 0.659150816 −1   0.228144395 0.4387690430.834567222 0.413629236 0.549081138 −1   0.45220778 0.5852384570.886623117 −0.103037336 0.838014042 −1   0.237802299 0.444914613−0.939531248 0.078411043 0.295691568 ZNF542_1 ZNF471_2 ZNF471_1 ZNF471_3ZNF132_2 coefs ZNF542 ′502 ZNF471 ′527 ZNF471 ′558 ZNF471 ′662 ZNF132′268   1   0.210023302 0.448781467 0.381358009 0.276869984 −0.079808848  1   0.096511033 −1.701659498 0.111027436 0.245615084 0.220719423  0.160992568   0.011755205 0.491015986 0.192253523 0.0118827880.004011962   1   0.075322076 0.362552655 0.179561947 0.3420976020.355650484   1   0.438561338 0.531490735 0.353436542 0.3516099630.275918493   1   0.322022075 0.502454502 0.314092655 0.1939765540.091921593   0.539110203   0.007214715 0.395108431 0.0919900710.143696932 −0.206562268   1   0.010241709 0.604521258 0.2988627640.238820541 0.200275323   0.228841426   0.010241709 −1.701659498−0.009542539 −3.291624259 0.120543333   1   0.477912258 0.2208910370.146563849 0.172234015 0.333161974   1   0.228185265 0.3810302580.097066702 0.160003836 0.380183404   0.086502196   0.116186493−1.701659498 −0.38775151 −0.122649173 −0.237228418   1   0.1948883330.592202857 0.357244014 0.299971432 0.053077803   0.022981588−0.13808099 −1.701659498 −0.060308843 −3.291624259 −0.186118168  0.199753156   0.15705091 0.495415416 0.260788035 0.227949271−0.034831828   0.640672506   0.120726984 −1.701659498 0.2150983610.210283458 0.183920043   0.488436741   0.05867361 −1.7016594980.02853219 0.007806062 −0.341493329   0.290813367   0.0632141010.449661352 0.10848912 0.248332902 0.433338064   0.93438076  0.043538641 0.359912998 0.034877978 −0.051985921 0.051033393   1  0.211536799 −1.701659498 −0.187224606 −0.012577568 0.482403904  0.177133374   0.264509192 0.375750943 0.046300397 −3.291624259−7.69E−05   0.363673626   0.073808579 0.258726128 0.171947002−0.049268103 −0.204517858   0.372869951 −3.407234345 0.5279711910.279825399 0.138261297 −0.406914449   0.180416799 −3.407234345−1.701659498 −0.136458301 −3.291624259 −0.406914449   1   1.3451459951.182606248 1.282459921 1.285180239 1.347189337   0.236974727−3.407234345 −1.701659498 −2.980640533 −0.383559643 −0.085942078  0.433173404   0.001160727 0.382790029 0.060261131 −0.069651734−0.110474998   0.037394649 −0.468023319 0.373111285 0.013302299−0.008500842 −0.257672518   1   0.455209804 0.5957224 0.4092794770.446733572 0.001967552   0.263636311 −3.407234345 −1.701659498−2.980640533 0.049932232 −5.066124854   1   0.721585263 0.521811990.325515074 0.344815419 0.791109815   1   0.060187107 0.3423152810.10848912 −0.046550286 0.071477493   1 −0.038190193 0.4197452340.317900128 0.173592923 −0.413047679   0.26169536 −3.4072343450.529730963 0.213829203 −0.096829908 −0.366026249   0.1799778  0.155537413 −1.701659498 −0.034925691 0.191258736 −5.066124854  0.898868481   0.278130664 0.655554636 0.5463485 0.405966311−0.192251398   0.042908076   0.079862567 0.491895872 0.2252516220.179028558 0.020367243   0.140898434   0.051106125 −1.701659498−0.243067541 −3.291624259 −5.066124854   0.140415448 −0.074514119−1.701659498 −2.980640533 0.154568201 −0.304693949   1   0.1282944690.220011151 −0.135189143 0.202130006 0.106232463   0.108835219−3.407234345 −1.701659498 −2.980640533 −3.291624259 0.026500473   1  0.526344159 −1.701659498 −0.140265774 0.233384906 0.603024094 −1  0.368940479 0.572845368 0.377550536 0.036343145 0.177786813 −1  0.31445459 0.503334388 0.298862764 0.181746375 0.132809793 −1  0.240293241 0.61683966 0.287440345 0.473911746 0.378138994 −1  0.134348456 0.407426833 0.248096459 0.279587802 −0.165674068 −1  0.424939866 0.570205711 0.39151127 0.305407067 0.523292104 −1  0.347751522 0.558767195 0.404202846 0.324431789 0.210497373 −1  0.325049069 0.280723273 0.102143332 0.295894706 −0.286294258 −1  0.577803055 0.540289593 0.434662629 0.505166646 0.881063855−0.27309104   1.379956424 0.847369747 0.820486547 0.7103618591.046661066 −1   1.806762557 1.486166858 1.711435198 1.7200310232.25286297 −1   0.343211032 0.614200002 0.400395374 0.4263499410.654134345 −0.675668601   0.455209804 0.547328679 0.4245093680.408684128 0.776798945 −1 −0.012460745 0.328237108 −0.031118218−0.007141934 0.077610723 −0.228886868   1.384496915 1.1518102441.269768345 1.328665317 1.330834057 −1   0.391642933 0.7549817350.631382061 −3.291624259 0.118498923 −0.253709663   0.7412607231.184366019 1.220271198 1.031064312 0.367916944 −1 −0.147161972−1.701659498 −0.187224606 −0.133520443 0.065344263 −1   0.5369386380.610680459 0.408010319 0.446733572 0.425160424 −1   0.188834345−1.701659498 0.259518877 0.237461632 0.480359494 −1   0.4219128720.484856786 0.344552438 0.318996154 0.692978135 −1   0.1903478420.604521258 0.31663097 0.270075441 −0.024609778 −1   0.0601871070.410946376 0.26332635 0.282305619 0.108276873 −1   0.0435386410.546448794 0.279825399 0.100211854 −0.063453568 −1   0.4431018290.402147518 0.429585999 0.274152167 0.055122213 −1   0.4461288220.451421124 0.211290888 0.308124884 0.220719423 −1   0.7745576550.410946376 0.221444149 0.145055841 0.717511055 −1   0.1101325050.549968337 0.243019828 −0.111777904 0.273874083 ZNF132_1 JAM2 MCIDAS_1MCIDAS_2 PDGFD_1 coefs ZNF132 ′415 JAM2 ′320 MCIDAS ′855 MCIDAS ′003PDGFD ′388   1   0.238896666 0.406419198 0.492859834 0.4164500630.046762915   1 −0.397730773 −2.423671354 0.320276073 0.121123394−0.124572283   0.160992568 −0.170552517 −2.423671354 −2.0522430390.091026409 −0.261640442   1   0.347202579 0.464995238 0.4857534440.245273459 0.426555938   1   0.120024323 0.545266109 0.7618874610.544362251 −0.027482338   1   0.006435195 0.297945048 0.5243309910.190722673 0.235231633   0.539110203 −0.693590827 0.2285215930.262409754 0.038356684 −0.53577676   1 −0.550944015 0.2870976330.044142056 −0.183608583 0.20382018   0.228841426 −0.3528234430.214419954 0.199467441 0.028951376 0.121008168   1   0.2996536410.447639374 0.58219731 0.501097835 0.423700352   1   0.267954350.317470395 0.572045325 0.252797705 0.266643087   0.086502196−0.487545432 0.333741518 0.359868819 0.213295412 −0.16169491   1  0.088325031 0.395571783 0.40961355 0.333683353 0.126719341  0.022981588 −1.155872162 −2.423671354 −2.052243039 0.019546069−0.669989332   0.199753156 −0.231309492 −2.423671354 0.2258626040.043999869 −0.17882843   0.640672506   0.407959554 0.3782159190.320276073 0.141815072 0.152419621   0.488436741 −0.410938811−0.105578787 0.311139286 −4.590935879 −0.375863908   0.290813367−0.049038566 0.15367443 −2.052243039 0.168149934 0.358021859  0.93438076 −0.614342598 0.187301416 −0.001541881 −0.078269134−0.624299946   1   0.415884377 0.216589437 0.588288502 0.2415113360.469389738   0.177133374   0.135873968 0.230691076 0.4431151030.15122038 0.132430514   0.363673626 −0.025264097 0.1308948590.318245676 0.072215793 −0.247362509   0.372869951 −0.2815000370.267572287 0.200482639 −0.042528964 −0.190250776   0.180416799−6.542110115 0.37930066 0.199467441 −0.245683615 −0.435831227   1  1.855560534 1.410889819 1.44105532 1.953277379 1.120463492  0.236974727 −0.71208208 −2.423671354 −2.052243039 −4.590935879−0.344452455   0.433173404   0.104174677 −0.128358358 0.297941704−0.078269134 0.292343366   0.037394649 −0.226026277 0.150420206−2.052243039 0.049643054 −0.104583177   1 −0.424146849 0.275165477−2.052243039 0.072215793 0.063896435   0.263636311 −6.542110115−0.08605344 −2.052243039 −0.591798947 −7.103626036   1   0.690611570.407503939 0.605546878 0.314872738 1.134741426   1 −0.0384721350.309877205 0.161905093 −0.241921492 0.295198953   1 −0.0146976670.199233573 0.256318562 −0.24004043 −0.407275361   0.26169536−0.878503361 0.122216927 0.038050865 −4.590935879 −0.415842121  0.1799778 −0.125645187 0.492113775 0.581182112 0.352493969−7.103626036   0.898868481   0.101533069 −2.423671354 0.416719940.02142713 −0.470098267   0.042908076 −0.281500037 −2.4236713540.402507159 −0.345379879 −0.235940162   0.140898434 −0.5324527620.177538743 −2.052243039 −0.040647902 −0.707111958   0.140415448−0.136211617 −2.423671354 0.160889894 0.143696133 −0.147416977   1  0.006435195 0.180792967 0.542604565 0.326159107 −0.304474242  0.108835219 −0.292066467 −2.423671354 0.051248446 −0.388644296−0.632866706   1   0.574380834 0.446554633 0.667473992 0.5989130370.812060135 −1   0.196630944 0.384724368 0.345656038 0.2189385970.363733032 −1   0.180781298 0.228521593 0.117236355 −0.1196524890.546490577 −1   0.471358137 0.249131681 0.483723047 −0.1497494740.483667671 −1 −0.022622489 0.469334204 0.406567954 0.145577195−0.007493231 −1   0.574380834 0.309877205 0.617729261 0.1324097640.466534151 −1   0.030209663 0.205742022 0.451236692 −0.003026671−0.010348818 −1   0.02228484 0.213335212 0.123327546 0.2377492130.078174368 −1   0.970621979 0.511639122 0.606562077 0.3111106140.689269909 −0.27309104   0.981188409 1.000857535 1.309079503 1.55073021.465989476 −1   2.840880179 1.538889315 1.185225275 1.657950712.031395631 −1   0.574380834 0.560452489 0.650215616 0.5612918050.777793095 −0.675668601   1.139684867 0.64072336 0.7202643190.572578175 0.892016561 −1 −0.178477339 0.079912009 0.1202819510.008259699 0.123863754 −0.228886868   1.945375193 1.1646535 1.0146719111.069178434 1.683014061 −1   0.064550562 0.462825755 0.5476805580.290418937 0.395144485 −0.253709663   0.79099266 0.7839092370.578136516 0.538719066 0.709259016 −1 −0.276216822 −2.4236713540.205558632 −0.010550917 −0.298763069 −1   0.36833544 0.4433004080.530422182 0.094788532 0.400855659 −1   0.36833544 0.2122504710.372051202 0.143696133 0.226664874 −1   0.761934976 0.5148933470.615698864 0.350612908 0.717825776 −1   0.283803995 0.4270292860.48169265 0.243392397 0.149564034 −1 −0.659249928 0.182962450.281698527 −0.371714741 0.118152581 −1 −0.241875922 0.1905556410.327382464 −0.001145609 −0.116005524 −1   0.149082006 0.2903518580.572045325 0.388234139 0.452256218 −1   0.347202579 0.4205208370.494890231 0.164387811 0.466534151 −1   1.055153423 0.5799778360.862392122 0.811472996 0.186686661 −1 −0.123003579 −2.4236713540.184239462 0.29418106 0.443689458 ST6GALNAC5 PDGFD_2 ST6GALNAC5ZNF492_1 CNRIP1_1 LONRF2_1 coefs PDGFD ′921 ′456 ZNF492 ′069 CNRIP1 ′272LONRF2 ′281   1   1.302789852 0.466406944 −0.240707793 −0.0641657950.182108844   1 −0.682617737 0.569041467 −0.17761095 −0.307307386−0.063816882   0.160992568 −0.682617737 0.705281983 −0.1505694460.093547669 −0.189987472   1 −0.682617737 0.569041467 0.1769332180.189928119 0.325387311   1   1.475318586 0.828806718 −0.1325417760.498783653 0.197078236   1 −0.682617737 −1.277471664 0.086794870.38268902 0.201355206   0.539110203 −0.682617737 −1.277471664−0.210661677 0.019071866 0.100846431   1 −0.682617737 0.3674055030.056748754 −0.149593922 0.090154008   0.228841426 −0.6826177370.246605578 0.194960887 0.161452077 −0.098032635   1   1.0966401330.76522781 0.480398989 0.178975795 0.534958799   1 −0.6826177370.792475913 0.065762589 0.119833246 0.338218218   0.086502196  1.281555546 −1.277471664 −0.327841529 −0.079499049 −0.207095348   1−0.682617737 0.638978265 0.149891714 0.369546231 0.333941249  0.022981588 −0.682617737 −1.277471664 −0.360892257 −3.744146627−0.585607118   0.199753156 −0.682617737 −1.277471664 −0.162587892−0.042261148 −5.029378066   0.640672506 −0.682617737 0.5862985990.23702545 −0.07073719 −0.22634171   0.488436741   1.209005001−1.277471664 0.017688804 −0.348926217 −0.160048688   0.290813367−0.682617737 0.638069995 0.110831763 −0.007213711 0.165000968  0.93438076   1.368262294 0.469131755 −0.57421968 −0.379592724−0.014631737   1 −0.682617737 −1.277471664 0.423311368 0.3169750760.498604562   0.177133374 −0.682617737 0.554509145 0.2430346730.222785091 0.154308545   0.363673626 −0.682617737 0.566316656−0.369906092 −0.186831823 −0.068093851   0.372869951 −0.682617737−1.277471664 −0.231693959 −0.06635626 −0.435913198   0.180416799−0.682617737 −1.277471664 −1.154109717 −2.41672497 −0.136525357   1−0.682617737 1.508192759 1.468916202 2.071537366 0.917747538  0.236974727 −0.682617737 −1.277471664 −0.330846141 −4.848140877−5.029378066   0.433173404 −0.682617737 0.542701634 0.056748754−0.243783907 0.263371258   0.037394649 −0.682617737 −1.2774716640.149891714 −0.362069006 −0.068093851   1 −0.682617737 0.6208128630.336177632 0.045357444 −0.311881093   0.263636311 −0.682617737−1.277471664 −7.154319065 −0.734448018 −5.029378066   1   1.438158551−1.277471664 1.072307472 0.660878047 0.791576948   1   1.2205069170.624445943 0.131864044 −0.048832542 −0.016770221   1 −0.6826177370.609913622 −0.204652454 −0.160546246 −0.269111401   0.26169536−0.682617737 −1.277471664 −7.154319065 0.207451837 −5.029378066  0.1799778 −0.682617737 −1.277471664 0.534481997 0.0628811620.607667275   0.898868481 −0.682617737 0.698015822 −0.0213711470.268784851 −0.040293551   0.042908076 −0.682617737 −1.277471664−0.339859976 −0.567972695 −0.326850485   0.140898434 −0.682617737−1.277471664 −0.571215068 −0.537306188 −0.442328652   0.140415448−0.682617737 −1.277471664 −0.144560222 −3.146149742 0.111538853   1−0.682617737 −1.277471664 −0.021371147 0.178975795 0.505020015  0.108835219 −0.682617737 −1.277471664 −0.318827695 -4.848140877−0.463713497   1 −0.682617737 −1.277471664 0.429320592 0.5601166670.757361195 −1   1.29571175 −1.277471664 0.092804093 −0.1364511330.107261884 −1 −0.682617737 0.76613608 0.393265253 −0.0948323020.220601567 −1 −0.682617737 −1.277471664 0.324159186 0.2600229920.415203663 −1   1.442582365 0.811549586 −0.108504883 0.4243078510.248401866 −1 −0.682617737 0.770677431 0.579551171 0.3804985550.637606059 −1   1.147071609 −1.277471664 −0.084467991 −0.2415934430.094430977 −1 −0.682617737 0.633528644 0.342186855 0.2162136970.081600069 −1 −0.682617737 0.821540557 0.792878594 0.6192592160.872839362 −0.27309104   2.042451502 1.427356719 1.2646026141.732015325 1.51224625 −1 −0.682617737 1.5981115 2.0127509 2.5293445052.128129808 −1   1.308983191 0.435525761 0.480398989 0.3169750760.733837865 −0.675668601 −0.682617737 0.837889419 0.7267771390.551354808 0.63332909 −1 −0.682617737 −1.277471664 0.083790259−0.06635626 −0.040293551 −0.228886868   1.923893295 1.3174560361.381782466 1.839348099 1.349721423 −1 −0.682617737 0.8569630910.315145351 0.343260654 0.259094289 −0.253709663   1.2355478831.01500209 0.964141455 1.026685666 0.729560896 −1   1.354106091−1.277471664 −0.048412651 −0.099213232 −0.076647789 −1 −0.6826177370.617179782 0.393265253 0.303832288 0.505020015 −1   1.2691688670.629895564 0.342186855 0.481259935 0.421619117 −1   1.2488193240.831531528 0.315145351 0.308213217 0.673960297 −1   1.3364108360.788842833 −0.168597115 0.371736696 0.188524298 −1   1.1338001680.567224926 −0.126532553 −0.134260668 −0.001800829 −1 −0.682617737−1.277471664 −0.105500272 −0.090451373 0.216324598 −1 −0.6826177370.717997765 0.390260641 0.411165062 0.513573954 −1 −0.6826177370.735254897 0.231016227 0.384879485 0.541374253 −1 −0.6826177371.01681863 0.231016227 1.147161228 0.703899081 −1   1.284209834−1.277471664 0.158905548 −0.103594161 −0.574914695 LONRF2_2 ADAMTS2_2ADAMTS2_1 ADAMTS2_3 ALK coefs LONRF2 ′387 ADAMTS2 ′254 ADAMTS2 ′284ADAMTS2 ′328 ALK ′434   1   0.816278101 −1.226540092 0.4792377160.308464228 1.224961864   1   0.707526931 0.608032885 0.3779728090.108720604 −0.67135809   0.160992568 −1.17952336 −1.226540092−1.402761026 −3.569757964 −0.67135809   1   0.644016248 0.7155112470.413319994 0.342021157 −0.67135809   1   1.028560383 0.8899568980.805960341 0.757487893 −0.67135809   1   0.704916903 0.7742109690.512674242 0.359598595 −0.67135809   0.539110203 −1.17952336 0.744447730.34931293 −0.159734825 −0.67135809   1 −1.17952336 −1.226540092−1.344485938 −0.001537876 1.290071562   0.228841426 −1.179523360.651850986 0.265243951 0.155061125 −0.67135809   1   0.8493384560.647717203 0.611073161 0.319649871 1.311232214   1   0.7884378010.675826929 0.455354484 0.27330935 −0.67135809   0.086502196  0.548315219 −1.226540092 0.357910894 0.155061125 1.30146576   1  0.564845397 0.690708548 −1.172526662 0.340423208 1.271352524  0.022981588 −1.17952336 −1.226540092 −1.818329276 −0.437777949−0.67135809   0.199753156   0.650976323 0.717164761 0.5795472940.310062177 −0.67135809   0.640672506 −1.17952336 0.763463132 0.445801190.290886789 −0.67135809   0.488436741 −1.17952336 0.6171272080.287216525 −0.153343029 −0.67135809   0.290813367   0.5709354630.735353407 0.491656997 0.316453973 −0.67135809   0.93438076 −1.179523360.666732606 0.444845861 0.169442666 −0.67135809   1 −0.8402197120.793226371 0.47350574 0.389959626 −0.67135809   0.177133374 −1.17952336−1.226540092 0.355044906 0.129493941 −0.67135809   0.363673626  0.538745116 −1.226540092 0.304412453 −0.142157387 −0.67135809  0.372869951 −1.17952336 −1.226540092 0.354089577 0.111916502−0.67135809   0.180416799   0.499594695 −1.226540092 −1.818329276−3.569757964 −0.67135809   1   0.880658793 1.437269791 1.3953985251.705071643 1.21519541   0.236974727   1.17952336 −1.226540092−1.818329276 −3.569757964 −0.67135809   0.433173404 −1.17952336−1.226540092 0.347402271 0.057586237 −0.67135809   0.037394649−1.17952336 −1.226540092 −1.818329276 −3.569757964 −0.67135809   1−1.17952336 0.671693146 0.532736158 −0.081435325 −0.67135809  0.263636311 −1.17952336 −1.226540092 −1.818329276 −3.569757964−0.67135809   1   0.846728428 0.660118553 0.491656997 0.4171247591.282746721   1   0.650106314 −1.226540092 0.401856042 −0.322725622−0.67135809   1   0.640536211 −1.226540092 0.567128013 0.254133962−0.67135809   0.26169536   0.519604911 −1.226540092 0.5365574750.284494993 −0.67135809   0.1799778   0.525694976 0.6138201810.533691487 0.266917554 −0.67135809   0.898868481 −1.179523360.719645031 0.407588018 −0.172518417 1.353553518   0.042908076  0.605735837 0.541065597 0.508852925 0.28928884 −0.67135809  0.140898434 −1.17952336 −1.226540092 −1.818329276 −3.569757964−0.67135809   0.140415448 −1.17952336 −1.226540092 −1.818329276−0.332313316 −0.67135809   1   0.771037614 −1.226540092 0.249958682−0.099012764 −0.67135809   0.108835219   0.456094228 0.611339911−1.818329276 −0.242828173 −0.67135809   1   0.865868634 −1.2265400920.373196163 0.410732963 −0.67135809 −1 −1.17952336 0.7179915170.575725977 0.27330935 1.293327047 −1 −0.466985698 0.5964582920.295814489 0.17903036 −0.67135809 −1   0.741457296 0.6750001720.399945384 0.287690891 −0.67135809 −1   0.838898344 0.7634631320.587189928 0.35160885 −0.67135809 −1   0.764077539 0.7957066410.609162503 0.4315063 −0.67135809 −1   0.920679223 −1.2265400920.530825499 0.139081635 1.257516713 −1 −1.17952336 0.6088596410.236584071 0.044802645 −0.67135809 −1   0.706656922 0.81554880.656928968 0.760683791 1.349484162 −0.27309104   1.3713440691.329791428 1.262607751 1.433420315 1.942796285 −1   1.8750794861.591046526 1.560670496 2.013475797 2.200793463 −1   0.5396151260.697322601 0.524138194 0.247742166 −0.67135809 −0.675668601  0.845858419 0.760982862 0.572859989 0.501816055 1.410524504 −1−1.17952336 0.765943402 0.286261196 −0.031898906 1.241239289−0.228886868   1.433114734 1.358727911 1.26451841 1.4813587841.862223034 −1   0.513514845 0.884996358 0.706606092 0.5673319641.322626411 −0.253709663   1.074670879 0.993301477 0.7906750720.69996173 1.619689409 −1 −1.17952336 0.583230186 0.3388043080.166246768 −0.67135809 −1   0.977229831 0.713030978 0.6034305270.370784238 −0.67135809 −1   0.684036679 0.742794216 0.4715950820.263721656 −0.67135809 −1   0.950259541 0.777517995 0.6502416630.568929913 1.333206737 −1   0.843248391 0.738660433 0.5824132820.319649871 −0.67135809 −1 −1.17952336 −1.226540092 0.244226706−0.234838428 −0.67135809 −1   0.717097034 −1.226540092 0.4582204720.107122655 −0.67135809 −1   0.689256735 −1.226540092 0.3426256250.242948319 −0.67135809 −1   0.864128615 0.785785562 0.5976985510.452279637 −0.67135809 −1   1.023340327 −1.226540092 0.2757525730.006451869 1.64085006 −1 −1.17952336 0.702283141 0.5164955590.334031412 −0.67135809 FGF14 DMRT1 CNRIP1_2 coefs FGF14 ′577 DMRT1 ′934CNRIP1 ′232   1 0.955300844 1.131612847 0.296394201   1 −0.93950462−0.701307401 0.101966839   0.160992568 0.846924723 −0.701307401−2.336804129   1 −0.93950462 −0.701307401 0.230086604   1 1.2964191271.516680127 0.443619546   1 0.974844079 −0.701307401 0.407656103  0.539110203 −0.93950462 1.17300758 0.203114022   1 −0.93950462−0.701307401 0.261554616   0.228841426 −0.93950462 1.1999622890.268297762   1 −0.93950462 −0.701307401 0.368321087   1 0.734106957−0.701307401 0.228962746   0.086502196 0.761645152 −0.7013074010.236829749   1 −0.93950462 −0.701307401 0.536899725   0.022981588−0.93950462 −0.701307401 −2.336804129   0.199753156 0.771416769−0.701307401 0.216600313   0.640672506 0.814944884 −0.7013074010.384055093   0.488436741 −0.93950462 −0.701307401 0.073870399  0.290813367 −0.93950462 −0.701307401 0.268297762   0.93438076−0.93950462 −0.701307401 0.027792238   1 0.960630817 −0.7013074010.3997891   0.177133374 −0.93950462 −0.701307401 0.092975978  0.363673626 0.769640112 −0.701307401 0.317747496   0.372869951−0.93950462 −0.701307401 0.225591173   0.180416799 −0.939504621.108508811 −2.336804129   1 1.728146955 1.533045486 1.467453809  0.236974727 −0.93950462 −0.701307401 −2.336804129   0.433173404−0.93950462 −0.701307401 0.284031768   0.037394649 −0.93950462−0.701307401 −2.336804129   1 0.834488119 −0.701307401 0.25031604  0.263636311 −0.93950462 −0.701307401 −2.336804129   1 0.798066635−0.701307401 0.540271298   1 −0.93950462 −0.701307401 −0.045258506   10.853143025 1.17300758 −2.336804129   0.26169536 −0.93950462−0.701307401 0.450362691   0.1799778 −0.93950462 1.108508811 0.412151533  0.898868481 −0.93950462 −0.701307401 0.412151533   0.042908076−0.93950462 −0.701307401 0.205361737   0.140898434 −0.93950462−0.701307401 −2.336804129   0.140415448 0.748320219 −0.701307401−2.336804129   1 0.726111997 −0.701307401 0.384055093   0.108835219−0.93950462 −0.701307401 −2.336804129   1 −0.93950462 1.124874170.598711893 −1 0.830046474 −0.701307401 0.314375923 −1 −0.93950462−0.701307401 0.078365829 −1 −0.93950462 −0.701307401 0.552633732 −10.867356287 1.072890087 0.532404295 −1 1.115200039 −0.7013074010.452610406 −1 0.632837467 1.202850294 0.307632777 −1 0.974844079−0.701307401 0.05476482 −1 0.946417555 −0.701307401 0.585225602−0.27309104 1.777893371 2.092355709 1.213461994 −1 1.8915994652.29451603 1.66750046 −1 0.822939843 −0.701307401 −2.336804129−0.675668601 0.790071676 −0.701307401 0.593092605 −1 −0.93950462−0.701307401 0.252563755 −0.228886868 1.602004256 1.9835742021.266283301 −1 1.124971657 1.209588971 0.542519013 −0.2537096631.057458663 −0.701307401 0.829102699 −1 1.010377234 −0.7013074010.017677519 −1 0.846924723 1.254834377 0.354834796 −1 1.0494637031.121023497 0.557129162 −1 −0.93950462 −0.701307401 0.488573849 −10.948194213 0.985287281 0.479582988 −1 −0.93950462 −0.701307401−2.336804129 −1 −0.93950462 −0.701307401 0.325614499 −1 0.225094519−0.701307401 0.389674381 −1 0.870909602 1.213439644 0.285155626 −1−0.93950462 1.568664209 0.829102699 −1 −0.93950462 −0.7013074010.224467316

Example 5. Various Individual Methylation Biomarkers are Each HighlyInformative

Evaluation of the performance of individual colorectal cancer DMRs fromamong the 28 colorectal cancer DMR panel reveal that various individualcolorectal cancer DMRs are sufficient for screening of colorectal cancer(See FIGS. 12-19). For selected colorectal cancer DMRs, FIGS. 12-19 showmethylation status of the indicated DMR in colorectal cancer samples andcontrol samples. Results are displayed as the MSRE-qPCR Ct valuesubtracted from 45 (i.e., 45−Ct value) for display purposes. Dataprovided in this Example, as well as data provided by the presentExamples cumulatively (including, e.g., FIGS. 5-9), demonstrate that foreach individual colorectal cancer DMR the methylation status signal issufficiently stable across subject groups to permit clinical screening.Results presented in FIGS. 12-19 therefore confirm that methylationmarkers of colorectal cancer provided herein can provide a robust signalfor screening of colorectal cancer. Moreover, those of skill in the artwill appreciate that the present disclosure provides methylationbiomarkers that are individually independently useful in screening forcolorectal cancer, and specifically that methylation biomarkers providedherein are useful both individually or in combination.

Example 6. Validation of Markers

Blood plasma samples were used to determine a minimal viable DMR panelfor the detection of colorectal cancer. It was found that themethylation status of DMRs was useful in distinguishing colorectalcancer not only from healthy subjects, but also from subjects sufferingwith other types of cancers (e.g., breast cancer, lung cancer).

Blood plasma samples from 215 subjects were analyzed using MSRE-qPCR andused as a training set to determine a minimal viable DMR panel forcolorectal cancer detection. FIG. 21 presents a detailed view of thecohort of subjects used for training an algorithm to detect colorectalcancer. The training set included 93 human subjects having beendiagnosed with colorectal cancer (CRC), 91 subjects having beendiagnosed as being healthy, and 31 subjects diagnosed has havingnon-advanced adenoma (NAA). The NAA+Healthy column is the sum of theHealthy and NAA columns. For the sake of calculating specificities, allpatients that did not have a colorectal cancer diagnosis were consideredto be controls. FIG. 21 also indicates the colorectal cancerdistribution. Colorectal cancer is classified as being localized oradvanced as determined by histology. The location of colorectal canceras determined by colonoscopy is either found to be in the proximal ordistal colon.

Selected DMRs that showed potential in colorectal cancer detection werefurther validated using an independent validation subject set (see FIG.22). Plasma samples of 774 human subjects were collected in Spain,Ukraine, the UK and the US. Of those 774 subjects, 152 subjects werediagnosed as having colorectal cancer (CRC). The control group included622 subjects. 148 subjects of the control group subjects hadnon-advanced adenoma, and 52 of the control group subjects had non-CRCcancer (i.e., breast cancer or lung cancer). FIG. 22 sets forth thenumber of males and females in each group, age range, and total numberof subjects. For those suffering with a cancer (i.e., colorectal,breast, or lung cancer), the cancer is classified as being localized oradvanced.

A random forest feature selection algorithm was implemented for featureranking. The algorithm utilized Monte-Carlo cross-validation over 50sub-setting iterations on the training set of FIG. 21 to rank thepre-selected markers according to their variance of importance (VIP) inoccurring in the 50 iterations. Markers with VIP>2 were further used forsupport-vector machine algorithm building on the training set.

It was found that 3-DMR (FIG. 23), 5-DMR (FIG. 24), and 6-DMR (FIG. 25)panels all performed well in assessing the colorectal cancer status ofsubjects of the validation set (FIG. 22). Tables 18, 19, and 20 (shownbelow) identify the markers included in each of the 3, 5, and 6 markerpanels, respectively.

TABLE 18 3 DMR Panel. DMR SEQ ID NO ZNF132 ′415 SEQ ID NO: 40 ADAMTS2′254 SEQ ID NO: 21 ADAMTS2 ′284 SEQ ID NO: 22

TABLE 19 5 DMR Panel. DMR SEQ ID ZNF132 ′415 SEQ ID NO: 40 ADAMTS2 ′254SEQ ID NO: 21 ADAMTS2 ′284 SEQ ID NO: 22 ZNF542 ′502 SEQ ID NO: 35LONRF2 ′281 SEQ ID NO: 19

TABLE 20 6 DMR Panel. DMR SEQ ID ZNF132 ′415 SEQ ID NO: 40 ADAMTS2 ′254SEQ ID NO: 21 ADAMTS2 ′284 SEQ ID NO: 22 ZNF542 ′502 SEQ ID NO: 35LONRF2 ′281 SEQ ID NO: 19 ZNF492 ′069 SEQ ID NO: 42

Furthermore, Table 21 (below) shows the accuracy, sensitivity,specificity and AUC of each of the 3-DMR (Table 18), 5-DMR (Table 19),and 6-DMR (Table 20) panels as previously presented.

TABLE 21 ROC Curve Analysis of Each Model 3 DMRs (2 Genes) 5 DMRs (4Genes) 6 DMRs (5 Genes) AUC 0.85 0.87 0.86 Sensitivity 0.58 0.60 0.69Specificity 0.89 0.90 0.87 Accuracy 0.83 0.84 0.83

The best performing algorithm used the 6 markers of Table 20 andresulted in 69% of the colorectal cancer cases being detected at 87%specificity with total AUC of 86%. Additionally, both 3-marker and5-marker panels performed well in classifying colorectal cancer. In the3 marker panel, 58% of the colorectal cancer cases were detected with aspecificity of 89% and AUC of 84.5%.

These findings are especially notable as high specificity was maintainedeven though subjects having cancer diagnoses other than colorectalcancer were included in the control group. This finding indicates thatthe markers in question are not generally indicative of cancer, but arespecifically and surprisingly important for detecting colorectal cancer.

In some embodiments, the present disclosure includes combinations ofDMRs in which each of the DMRs is, includes all of, includes a portionof, or is present in a gene identified in Table 22. In some embodiments,the present disclosure includes combinations of DMRs in which each ofthe DMRs is, includes all of, includes a portion of, or is present in agene identified in Table 23. In some embodiments, the present disclosureincludes combinations of DMRs in which each of the DMRs is, includes allof, includes a portion of, or is present in a gene identified in Table24.

In some particular embodiments, a colorectal cancer methylationbiomarker includes two DMRs, each of which is, includes all of, includesa portion of, or is present in a different gene identified in Table 22.In some particular embodiments, a colorectal cancer methylationbiomarker includes four DMRs, each of which is, includes all of,includes a portion of, or is present in a different gene identified inTable 23. In some particular embodiments, a colorectal cancermethylation biomarker includes five DMRs, each of which is, includes allof, includes a portion of, or is present in a different gene identifiedin Table 24.

In various embodiments, a colorectal cancer methylation biomarkerincludes two or more DMRs that include all of, include a portion of, orare present in the same gene of Table 22. In various embodiments, acolorectal cancer methylation biomarker includes two or more DMRs thatinclude all of, include a portion of, or are present in the same gene ofTable 23. In various embodiments, a colorectal cancer methylationbiomarker includes two or more DMRs that include all of, include aportion of, or are present in the same gene of Table 24.

TABLE 22 Genes of DMR Panel of Table 18. ZNF132 ADAMTS2

TABLE 23 Genes of DMR Panel of Table 19. ZNF132 ADAMTS2 ZNF542 LONRF2

TABLE 24 Genes of DMR Panel of Table 20 ZNF132 ADAMTS2 ZNF542 LONRF2ZNF492

Example 7. Various Individual Methylation Biomarkers are Each HighlyInformative

Evaluation of the performance of individual colorectal cancer DMRs fromamong the colorectal cancer DMR panel (e.g., as set forth in Example 6)reveal that various individual colorectal cancer DMRs are sufficient forscreening of colorectal cancer from not only healthy individuals butalso from individuals suffering from other cancers. Univariate analysisover methylation status of each of the methylation markers as shown inTable 25 below shows a number of markers with high individual accuracyas indicated by p-values being less than 0.001.

TABLE 25 T-Test P-Values Between CRC and Control Groups in ValidationSet Gene Reference P-value of Name Student's t-test ZNF132 ′415 4.52E−25ADAMTS2 ′254 8.00E−29 ADAMTS2 ′284 1.62E−21 ZNF542 ′502 1.76E−37 LONRF2′281 9.26E−38 ZNF492 ′069 1.18E−25

For the selected colorectal cancer DMRs of Table 25, FIGS. 26-31 furthershow methylation status of the indicated DMR in colorectal cancersamples and control samples (i.e., of the validation group of FIG. 22).In this instance, control samples comprise healthy subjects, subjectshaving non-advanced adenomas, subjects having breast cancer, andsubjects having lung cancer (see, FIG. 22). Results are displayed as theMSRE-qPCR Ct value subtracted from 45 (i.e., 45−Ct value) for displaypurposes. Data provided in this Example, as well as data provided by thepresent Examples cumulatively (including, e.g., FIGS. 23-25),demonstrate that for each individual colorectal cancer DMR themethylation status signal is sufficiently and surprisingly stable acrosssubject groups to permit clinical screening, particularly indiscriminating colorectal cancer from other forms of cancer. Resultspresented in FIGS. 23-25 therefore further confirm that methylationmarkers of colorectal cancer provided herein can provide a robust signalfor screening of colorectal cancer. Moreover, those of skill in the artwill appreciate that the present disclosure provides that thesemethylation biomarkers are individually useful in screening forcolorectal cancer, and specifically that methylation biomarkers providedherein are useful both individually or in combination.

SEQUENCES (ZNF132) (see Table 1)SEQ ID NO: 1 >ZNF132 chr19, bp 58439728 to 58440994 of hg38(DMRT1) (see Table 1)SEQ ID NO: 2 >DMRT1 chr9, bp 841340 to 968090 of hg38(ALK) (see Table 1)SEQ ID NO: 3 >ALK chr2, bp 29193215 to 29922286 of hg38(JAM2) (see Table 1)SEQ ID NO: 4 >JAM2 chr21, bp 25637848 to 25714704 of hg38(FGF14) (see Table 1)SEQ ID NO: 5 >FGF14 chr13, bp 101919879 to 102403137 of hg38(MCIDAS) (see Table 1)SEQ ID NO: 6 >MCIDAS chr5, bp 55220951 to 55221051 of hg38(ST6GALNAC5) (see Table 1)SEQ ID NO: 7 >ST6GALNAC5 chr1, bp 76866255 to 77063388 of hg38(LONRF2) (see Table 1)SEQ ID NO: 8 >LONRF2 chr2, bp 100285667 to 100323015 of hg38(PDGFD) (see Table 1)SEQ ID NO: 9 >PDGFD chr11, bp 104163499 to 104164026 of hg38(GSG1L) (see Table 1)SEQ ID NO: 10 >GSG1L chr16, bp 27920615 to 28064275 of hg38(ZNF492) (see Table 1)SEQ ID NO: 11 >ZNF492 chr19, bp 22633051 to 22666433 of hg38(ZNF568) (see Table 1)SEQ ID NO: 12 >ZNF568 chr19, bp 36916312 to 36943940 of hg38(ADAMTS2) (see Table 1)SEQ ID NO: 13 >ADAMTS2 chr5, bp 179118114 to 179344392 of hg38(ZNF542) (see Table 1)SEQ ID NO: 14 >ZNF542 chr19, bp 56367838 to 56370986 of hg38(ZNF471) (see Table 1)SEQ ID NO: 15 >ZNF471 chr19, bp 56507245 to 56508589 of hg38(CNRIP1) (see Table 1)SEQ ID NO: 16 >CNRIP1 chr2, bp 68293114 to 68320928 of hg38(see Table 7) SEQ ID NO: 17CCTCCTCACCCATCATCAGCGCCCGCGGCTTTGGGTGGCCGACCAGAGGGCGGCCGGAAAGCACCTCGGTGCCCCGCGACCCTCCGAACAGAGGCGGCGGGA GGTACC (see Table 7)SEQ ID NO: 18 GCGTGCTGGGTTTAATCTTCACCTCAACCTTGTAGGAGGAGCCGGTGAGCAGCTTGATGGTGCGGTTCTGGCCGAAGCGCTGCCCGTCCACCTTGTAAAAGA CCGGGCCGT(see Tables 7, 19, and 20) SEQ ID NO: 19AGGAAGCAAAGTGACCCCTAAGCCTAGACAAAGCTCTCGAAAGCCCAAAGCCTCGGGCCCACCGGCCAGCTCCCCACCCCGCTGCTGGGCCGGACAGGTGTA GGGGAGGCGGACC(see Table 7) SEQ ID NO: 20CTCTCAGTCCCGCCGGCTTAGGTAACCCAGGTCGCTGCGGTAACGCAGTGACCGCGCTCCAGGTCCGCGTCTCTTGC (see Tables 7, 18, 19, and 20) SEQ ID NO: 21CCACTGCGAAGGGAAGGGGCATTCCGCCAGGCGACCCCAGAAGCCAGCCTGCACCTCCCCGGCTTTCCTGCAACCGGGAAGGGGCGTTAACAGGG(see Tables 7, 18, 19, and 20) SEQ ID NO: 22GCGACCCCAGAAGCCAGCCTGCACCTCCCCGGCTTTCCTGCAACCGGGAAGGGGCGTTAACAGGGCCACCACTCCGGGGCTCCGCCACTCCCCAGCCGTT (see Table 7)SEQ ID NO: 23 CAACGGAAACTTCCCGCGCTACGGCGGCTCCAACGGGCCGCTTCCGCCGCATTGCGTAGCGAAGCCCCCGGCGAG (see Table 7) SEQ ID NO: 24CAAAGCGTCTGGGGCGCTAGTGGGGGCGGCCAGCGGCTCGAGCGCCGGGGGCAGCAGCAGAGGAGGCGGCTCCGGCTCCGGGGCGTCGGACCTGGGTGCCGG GAGCAAGAAGT(see Table 7) SEQ ID NO: 25CGCTCAGCCGCTCTCCTCTTCTCTCTCCCGCCCGCCCGCAGCGCCATGGTC TGGCAGTGTGTTTAGCGCT(see Table 7) SEQ ID NO: 26GGGTTCGGAGCGTGCAAAAGGTGACCTAGGCGCGCTACGCACCACGCACTCAGCGGTACTCTCCTCTCCCGGGCCCCCACGGGTCCCGATGCTGGGCGGGGA TGCACTGAACTGTTC(see Table 7) SEQ ID NO: 27GCGCCCCACTTACATCCAGCACCGAGGCCAGGTGCCGGGTTCGGCTGGCGAGTTCCTTCAGCTGCACGTTCCGCTCCTTGAGCGAGGCGATCTCCTCCTGTT TCTGGGTCAATGTCACGT(see Table 7) SEQ ID NO: 28AACGTCTATCACCCAGGGAAAGCTACTCTTGACTCCTTCCACCTATCAAAATTGCCTAAGAAAGGTTGAGTCTGACCAAGGGGCGGCGCAGCTTGCAACTTT CGCCAACTCCGGGA(see Table 7) SEQ ID NO: 29GGTGCATTTGGGATCAGCGACTAGAGACAGCGTCGCTCCAAGAAAAAGCCGGGTTCTGCTCCCGGGACCGACGCCGCGCCGCCCTGCGCTCTCGCCGCCTGCGCTCGCCCTGCGCTGGCCCGGGTCGCTGTGCTAATC (see Table 7) SEQ ID NO: 30CCGAAAGAAATCCGAGCCAGGGTGAGGGTCTGAGACGCAAGGAGAATCCCAGGCAAGGCGCTCCTGAGAAAAGATCCCCACGGCGGACGTGGGGCAACAAAA CC (see Table 7)SEQ ID NO: 31 CGAGAGAGGGGAAGGGGCTGGTTGGAACCGGTGGCAAGAGGCTGTGGCGGGACTCAGGCCTCCCCGCAGTCGGCTCCACAATCTGCGCCCCAAGTTCG (see Table 7)SEQ ID NO: 32 GCCCAAGCCTCACCCTCACACAGGAAAGCAGATGTGTTCTGGCCGGAAGTTGAGTGGGGCCGCGGGGCCTGCTGGGAGGTGTTGTCCTCGGAAACGTCGCTG GCGCGGAGGGATGGTTCG(see Table 7) SEQ ID NO: 33GGTCGCCTTCACCCAGCATCTCAGAAACTGCGCGCGGGATGAACATTCGGGTGTTTCCGGCAGGTGACGCTG (see Table 7) SEQ ID NO: 34CCAGAGGCCCAGGGATCCGTTCAGGTCAGCGCTGGCGTCCGGGCCTGAGTTTGGAGGTGGCGGGTGCCTTACAAGAATGCTCGCGT (see Tables 7, 19, and 20)SEQ ID NO: 35 GGGAGGAGTGGGCGGCTGAATGGCCAGAGGCCCAGGGATCCGTTCAGGTCAGCGCTGGCGTCCGGGCCTGAGTTTGGAGGTGGCGGGTGC (see Table 7) SEQ ID NO: 36CCCCACGCGTACTCACACCGAAGGCTCAGCCGTCGCGCGTTTCCCTCCCAGGCCCCAGGAACTAGTAACTAGGGACGCTTCTGGTCTCTAGGCGAGGAGAGGGGGAGAGCGCAATCTTTGCGCCTGCGCACACTCCTGCTCTTACCCGC (see Table 7)SEQ ID NO: 37 GTCGCGCGTTTCCCTCCCAGGCCCCAGGAACTAGTAACTAGGGACGCTTCTGGTCTCTAGGCGAGGAGAGGGGGAGAGCGCAATCTTTGCGCCTGCGCACAC TCCTGCTCTTACCCGC(see Table 7) SEQ ID NO: 38CTGCTCTTACCCGCCGGAACCCTGGGCCACGCCCGGCTCGCGTAATCACGCACTGCGCAGGCACCGCCCGCTCTGCTCTAAGGTCCCTC (see Table 7) SEQ ID NO: 39CTACTGCTAGGTCGTTGCCAAGGTGATTGAGGAATGGCGTTTATTGCGTCGCTGCTCAGGCAACGCAAACTACATTATCCAGAAGGACCCTCGCGGTGCCTCAGGGCTGGCCATTGGCAGCCGAGGAGACAGGCACTTCCGGGCGGAGTGTAA GACGCTGGCCAATCA(see Tables 7, 18, 19, and 20) SEQ ID NO: 40GTGTAAGACGCTGGCCAATCACAGCCTGGCAGCGGGACTTCCGTCGTCGTCCTCGGACCATCACTTTGGCATTTCTCGATTTTGTCTGCTTCTGAAGGGACC GCGTTGT(see Table 7) SEQ ID NO: 41CCGCGTGGTCTGGGCTCTGTAGCGTCCCAGCTGAGCCGGCGATATGCAGCGCACTTGTGGGGCGGAGGTGGAGGGAATTC (see Tables 7 and 20) SEQ ID NO: 42CAACGTTAAAGGCAAACACCTTCTGCGGTGTGCTTGGCTCAGCTCAGGCAGGAAGCCCTGCCTGAAAAGGCTGCACCTTCGGCTGTCACTCTGTCCTCATTC GGCC (see Table 7)SEQ ID NO: 43 GCCGGTGAGCAGCTTGATGGTGCGGTTCTGGCCGAAGCGCTGCCCGTCCACCTTGTAAAAGACCGGGCCGT (see Table 7) SEQ ID NO: 44CGGGAAGGGGCGTTAACAGGGCCACCACTCCGGGGCTCCGCCACTCCCCAGCCGTTCCCTCCTCCGGAGACCTTGCCTGCCAAGA (see Table 13) SEQ ID NO: 45CCTCCTCACCCATCATCAGCGCCC (see Table 13) SEQ ID NO: 46GGTACCTCCCGCCGCCTCTGTTC (see Table 13) SEQ ID NO: 47GCGTGCTGGGTTTAATCTTCACCTCAA (see Table 13) SEQ ID NO: 48ACGGCCCGGTCTTTTACAAGGTGG (see Table 13) SEQ ID NO: 49AGGAAGCAAAGTGACCCCTAAGCCT (see Table 13) SEQ ID NO: 50GGTCCGCCTCCCCTACACCT (see Table 13) SEQ ID NO: 51CTCTCAGTCCCGCCGGCTTAGGTA (see Table 13) SEQ ID NO: 52GCAAGAGACGCGGACCTGGAGC (see Table 13) SEQ ID NO: 53CCACTGCGAAGGGAAGGGGCA (see Table 13) SEQ ID NO: 54CCCTGTTAACGCCCCTTCCCGGTT (see Table 13) SEQ ID NO: 55GCGACCCCAGAAGCCAGCCT (see Table 13) SEQ ID NO: 56 AACGGCTGGGGAGTGGCGGA(see Table 13) SEQ ID NO: 57 CAACGGAAACTTCCCGCGCTAC (see Table 13)SEQ ID NO: 58 CTCGCCGGGGGCTTCGCTAC (see Table 13) SEQ ID NO: 59CAAAGCGTCTGGGGCGCTAGT (see Table 13) SEQ ID NO: 60ACTTCTTGCTCCCGGCACCCAGGTC (see Table 13) SEQ ID NO: 61CGCTCAGCCGCTCTCCTCTTCTCT (see Table 13) SEQ ID NO: 62AGCGCTAAACACACTGCCAGACCA (see Table 13) SEQ ID NO: 63GGGTTCGGAGCGTGCAAAAGGTGA (see Table 13) SEQ ID NO: 64GAACAGTTCAGTGCATCCCCGCCC (see Table 13) SEQ ID NO: 65GCGCCCCACTTACATCCAGCACC (see Table 13) SEQ ID NO: 66ACGTGACATTGACCCAGAAACAGGAGGA (see Table 13) SEQ ID NO: 67AACGTCTATCACCCAGGGAAAGCT (see Table 13) SEQ ID NO: 68TCCCGGAGTTGGCGAAAGTTGCAA (see Table 13) SEQ ID NO: 69GGTGCATTTGGGATCAGCGACTAGAGAC (see Table 13) SEQ ID NO: 70GATTAGCACAGCGACCCGGGCCAG (see Table 13) SEQ ID NO: 71CCGAAAGAAATCCGAGCCAGGGTGA (see Table 13) SEQ ID NO: 72GGTTTTGTTGCCCCACGTCC (see Table 13) SEQ ID NO: 73CGAGAGAGGGGAAGGGGCTGGTTG (see Table 13) SEQ ID NO: 74CGAACTTGGGGCGCAGATTGTGG (see Table 13) SEQ ID NO: 75GCCCAAGCCTCACCCTCACACAG (see Table 13) SEQ ID NO: 76CGAACCATCCCTCCGCGCCA (see Table 13) SEQ ID NO: 77GGTCGCCTTCACCCAGCATCTCAG (see Table 13) SEQ ID NO: 78CAGCGTCACCTGCCGGAAACACC (see Table 13) SEQ ID NO: 79CCAGAGGCCCAGGGATCCGTTCAG (see Table 13) SEQ ID NO: 80ACGCGAGCATTCTTGTAAGGCACCC (see Table 13) SEQ ID NO: 81GGGAGGAGTGGGCGGCTGAATGG (see Table 13) SEQ ID NO: 82GCACCCGCCACCTCCAAACTCAG (see Table 13) SEQ ID NO: 83CCCCACGCGTACTCACACCGAAG (see Table 13) SEQ ID NO: 84GCGGGTAAGAGCAGGAGTGTG (see Table 13) SEQ ID NO: 85 GTCGCGCGTTTCCCTCCCAG(see Table 13) SEQ ID NO: 86 GCGGGTAAGAGCAGGAGTGTG (see Table 13)SEQ ID NO: 87 CTGCTCTTACCCGCCGGAACCCTG (see Table 13) SEQ ID NO: 88GAGGGACCTTAGAGCAGAGCGGGC (see Table 13) SEQ ID NO: 89CTACTGCTAGGTCGTTGCCAAGG (see Table 13) SEQ ID NO: 90TGATTGGCCAGCGTCTTACACTCCG (see Table 13) SEQ ID NO: 91GTGTAAGACGCTGGCCAATCACA (see Table 13) SEQ ID NO: 92ACAACGCGGTCCCTTCAGAAGCAG (see Table 13) SEQ ID NO: 93CCGCGTGGTCTGGGCTCTGTAG (see Table 13) SEQ ID NO: 94GAATTCCCTCCACCTCCGCCCCAC (see Table 13) SEQ ID NO: 95CAACGTTAAAGGCAAACACCTTCTGC (see Table 13) SEQ ID NO: 96GGCCGAATGAGGACAGAGTGACAG (see Table 13) SEQ ID NO: 97GCCGGTGAGCAGCTTGATGGT (see Table 13) SEQ ID NO: 98 ACGGCCCGGTCTTTTACAAGG(see Table 13) SEQ ID NO: 99 CGGGAAGGGGCGTTAACAGGGC (see Table 13)SEQ ID NO: 100 TCTTGGCAGGCAAGGTCTCCGGAG

Other Embodiments

While we have described a number of embodiments, it is apparent that ourbasic disclosure and examples may provide other embodiments that utilizeor are encompassed by the compositions and methods described herein.Therefore, it will be appreciated that the scope of is to be defined bythat which may be understood from the disclosure and the appended claimsrather than by the specific embodiments that have been represented byway of example.

All references cited herein are hereby incorporated by reference.

What is claimed is:
 1. A method of detecting (e.g., screening for)colorectal cancer, the method comprising: determining a methylationstatus for each of the following, in deoxyribonucleic acid (DNA) of ahuman subject: (a) a methylation locus within gene ZNF132; (b) a firstmethylation locus within gene ADAMTS2; and (c) a second methylationlocus within gene ADAMTS2; and diagnosing colorectal cancer in the humansubject based on said determined methylation statuses.
 2. The method ofclaim 1, further comprising determining a methylation status for each ofthe following, in the DNA of the human subject: (d) a methylation locuswithin gene ZNF542; and (e) a methylation locus within gene LONRF2. 3.The method of claim 2, further comprising determining a methylationstatus for a methylation locus within gene ZNF492 in the DNA of thehuman subject.
 4. The method of claim 1, wherein the methylation locuswithin gene ZNF132 comprises ZNF132 '415 (SEQ ID NO: 40).
 5. The methodof claim 4, wherein the first methylation locus within gene ADAMTS2comprises ADAMTS2 '254 (SEQ ID NO: 21).
 6. The method of claim 5,wherein the second methylation locus within gene ADAMTS2 comprisesADAMTS2 '284 (SEQ ID NO: 22).
 7. The method of claim 2, wherein themethylation locus within gene ZNF542 comprises ZNF542 '502 (SEQ ID NO:35).
 8. The method of claim 2, wherein the methylation locus within geneLONRF2 comprises LONRF2 '281 (SEQ ID NO: 19).
 9. The method of claim 3,wherein the methylation locus within gene ZNF492 comprises ZNF492 '069(SEQ ID NO: 42).
 10. The method of claim 1, wherein the DNA is isolatedfrom blood or plasma of the human subject.
 11. The method of claim 1,wherein the DNA is cell-free DNA of the human subject.
 12. The method ofclaim 1, wherein methylation status is determined using quantitativepolymerase chain reaction (qPCR).
 13. A kit for use in colorectal cancerdetection (e.g., screening), the kit comprising: (a) an oligonucleotideprimer pair for amplification of a methylation locus within gene ZNF132;(b) an oligonucleotide primer pair for amplification of a firstmethylation locus within gene ADAMTS2; and (c) an oligonucleotide primerpair for amplification of a second methylation locus within geneADAMTS2.
 14. The kit of claim 13, further comprising: (d) anoligonucleotide primer pair for amplification of a methylation locuswithin gene ZNF542; and (e) an oligonucleotide primer pair foramplification of a methylation locus within gene LONRF2.
 15. The kit ofclaim 14, further comprising: (f) an oligonucleotide primer pair foramplification of a methylation locus within gene ZNF492.
 16. The kit ofclaim 13, wherein (a) is an oligonucleotide primer pair foramplification of ZNF132 '415 (primer pair SEQ ID NO: 91 and SEQ ID NO:92).
 17. The kit of claim 16, wherein (b) is an oligonucleotide primerpair for amplification of ADAMTS2 '254 (primer pair SEQ ID NO: 53 andSEQ ID NO: 54).
 18. The kit of claim 17, wherein (c) is anoligonucleotide primer pair for amplification of ADAMTS2 '284 (primerpair SEQ ID NO: 55 and SEQ ID NO: 56).
 19. The kit of claim 14, wherein(d) is an oligonucleotide primer pair for amplification of ZNF542 '502(primer pair SEQ ID NO: 81 and SEQ ID NO: 82).
 20. The kit of claim 14,wherein (e) is an oligonucleotide primer pair for amplification ofLONRF2 '281 (primer pair SEQ ID NO: 49 and SEQ ID NO: 50).
 21. The kitof claim 15, wherein (f) is an oligonucleotide primer pair foramplification of ZNF492 '069 (primer pair SEQ ID NO: 95 and SEQ ID NO:96).
 22. A diagnostic qPCR reaction for detection (e.g., screening) ofcolorectal cancer, the diagnostic qPCR reaction including: (a) humanDNA; (b) a polymerase; (c) an oligonucleotide primer pair foramplification of a methylation locus within gene ZNF132; (d) anoligonucleotide primer pair for amplification of a first methylationlocus within gene ADAMTS2; (e) an oligonucleotide primer pair foramplification of a second methylation locus within gene ADAMTS2; and (f)optionally, at least one methylation sensitive restriction enzyme. 23.The reaction of claim 22, further comprising: (g) an oligonucleotideprimer pair for amplification of a methylation locus within gene ZNF542;and (h) an oligonucleotide primer pair for amplification of amethylation locus within gene LONRF2.
 24. The reaction of claim 23,further comprising: (i) an oligonucleotide primer pair for amplificationof a methylation locus within gene ZNF492.
 25. The reaction of claim 22,wherein (c) is an oligonucleotide primer pair for amplification ofZNF132 '415 (primer pair SEQ ID NO: 91 and SEQ ID NO: 92).
 26. Thereaction of claim 25, wherein (d) is an oligonucleotide primer pair foramplification of ADAMTS2 '254 (primer pair SEQ ID NO: 53 and SEQ ID NO:54).
 27. The reaction of claim 26, wherein (e) is an oligonucleotideprimer pair for amplification of ADAMTS2 '284 (primer pair SEQ ID NO: 55and SEQ ID NO: 56).
 28. The reaction of claim 23, wherein (g) is anoligonucleotide primer pair for amplification of ZNF542 '502 (primerpair SEQ ID NO: 81 and SEQ ID NO: 82).
 29. The reaction of claim 23,wherein (h) is an oligonucleotide primer pair for amplification ofLONRF2 '281 (primer pair SEQ ID NO: 49 and SEQ ID NO: 50).
 30. Thereaction of claim 24, wherein (i) is an oligonucleotide primer pair foramplification of ZNF492 '069 (primer pair SEQ ID NO: 95 and SEQ ID NO:96).